METHODS FOR PRODUCTION OF FUCOSYLATED OLIGOSACCHARIDES IN RECOMBINANT CELL CULTURE

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on Aug. 11, 2023, is named 081906-1390975_242010US_SL.xml and is 175,778 bytes in size.

BACKGROUND OF THE INVENTION

Human milk oligosaccharides (HMOs) are a class of over 200 compounds present at 20-23 g/L in colostrum and 12-14 g/L in mature milk (Chen, 2015; Smilowitz et al., 2014; Wiciński et al., 2020; Yu and Chen, 2019). Unlike their common precursor lactose, HMOs are indigestible by human infants and instead improve neonatal health by serving as effective antimicrobials and antivirals, prebiotics, and regulators of inflammatory immune cell-response cascades (Ayechu-Muruzabal et al., 2018; Ballard and Morrow, 2013; Kulinich and Liu, 2016; Rudloff and Kunz, 2012; Triantis et al., 2018; Wicinski et al., 2020). These and other potential benefits of HMOs make them attractive targets of study for preventing or treating diseases in both children and adults (Wiciński et al., 2020). The bioactive properties of HMOs have motivated efforts to define mechanistic effects of individual compounds (Berger et al., 2020; Bode, 2012; Borewicz et al., 2020; Hegar et al., 2019), but the sources of HMOs are limited and their large-scale isolation for such studies is exceedingly difficult. While production of individual HMOs using in vitro enzymatic reactions has been successful (Ågoston et al., 2019; Bai et al., 2019; Bandara et al., 2019, 2020; McArthur et al., 2019; Xiao et al., 2016; Yu et al., 2017; Zhao et al., 2016), these methods require supplementation of stoichiometric amounts of ATP and other cofactors that increase the production cost and may complicate the purification process of the oligosaccharide products.

Microbial production is a viable alternative method to produce HMOs. Whole cell biocatalysts are self-maintaining systems and do not require an exogenous supply of expensive cofactors. Enzymatic reactions in cells can also achieve high regio- and stereo-specific production of structurally complex molecules. Several simple HMOs including 2′-FL, 3-FL, lacto-N-triose II, lacto-N-tetraose (LNT), and lacto-N-neotraose (LNnT) have been produced in engineered microorganisms (Baumgartner et al., 2014; Choi et al., 2019; Dong et al., 2019; Huang et al., 2017; Liu et al., 2020; Yu et al., 2018). Linear HMO backbones such as lactose, LNT, and LNnT can be glycosylated at multiple sites with fucose and sialic acid to further produce HMOs of higher structural complexity. While in vitro enzymatic synthesis can construct these decorated HMOs by strategically producing each intermediate HMO structure in individual reaction systems, microbial production of multi-glycosylated HMOs in a microbial host has not been demonstrated.

Tetrasaccharide lactodifucotetraose (LDFT) is one of the most abundant fucosylated HMOs and is produced at an average of 0.43 g/L over the first year of lactation by secretory mothers (Chaturvedi et al., 2001). Its structure consists of a core lactose unit that is fucosylated at the C2′ and C3 positions. Studies have shown that LDFT is effective in preventing Campylobacter jejuni-associated diarrhea and suppressing platelet-induced inflammatory processes in neonates (Newburg et al., 2016; Orczyk-Pawilowicz and Lis-Kuberka, 2020). Its activity as a gastrointestinal and immunological modulator has motivated further research into its potential therapeutic applications. However, the high cost and limited availability of LDFT in the market ($140/mg, Biosynth Carbosynth; €11,000/g Elicityl) are barriers to these biological studies.

BRIEF SUMMARY OF THE INVENTION

Provided herein are recombinants cell for production of oligosaccharide products such as difucosylated oligosaccharides. The cells include: a polynucleotide encoding a first glycosyltransferase polypeptide having a first substrate selectivity and a polynucleotide encoding a second glycosyltransferase polypeptide having a second substrate selectivity, and may further include one or more polynucleotides selected from the group consisting of: a polynucleotide encoding a nucleotide sugar pyrophosphorylase polypeptide, a monosaccharide transporter polypeptide, and an oligosaccharide transporter polypeptide.

In some embodiments, the cells include:

- a polynucleotide encoding a first fucosyltransferase polypeptide having a first substrate specificity (e.g., an α1-2-fucosyltransferase polypeptide),
- a polynucleotide encoding a second fucosyltransferase polypeptide having a second substrate specificity (e.g., an α1-3-fucosyltransferase polypeptide), and
- optionally one more polynucleotides encoding a nucleotide sugar pyrophosphorylase polypeptide, a lactose transporter polypeptide, and a polynucleotide encoding an L-fucose transporter polypeptide.

Also provided herein are methods for producing difucosylated oligosaccharides and other oligosaccharide products. In some embodiments, the methods include culturing a recombinant cell as described herein in a cell culture medium comprising L-fucose, an oligosaccharide acceptor, and a carbon source; wherein the cell is cultured under conditions in which a first fucosyltransferase polypeptide, a second fucosyltransferase polypeptide, a nucleotide sugar pyrophosphorylase polypeptide, a lactose transporter polypeptide, and an L-fucose transporter polypeptide are expressed and the oligosaccharide acceptor is converted to the difucosylated oligosaccharide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a summary of various pathway design aspects for LDFT production in E. coli according to the present disclosure. L-Fucose (triangles) and lactose (glucose moiety, diagonal striped circles; galactose moiety, filled circles) are transported into the cytosol through sugar transporters FucP and LacY, respectively. The fucU and lacZ genes are deleted to prevent substrate assimilation into central carbon metabolism. Fucose is converted to donor substrate GDP-fucose by Fkp. WbgL glycosylates lactose at the C2′ position with GDP-fucose to form 2′-FL. Hp3/4FT glycosylates 2′-FL at the C3 position with GDP-fucose to form LDFT. G-6-P: glucose-6-phosphate, DHAP: dihydroxyacetone phosphate, PTS: phosphotransferase system, SetA: sugar efflux transporter A.

FIG. 2A shows cell growth and protein expression resulting from modifications in E. coli B-strains for LDFT production. Growth of BL21 Star (DE3) and ΔlacZ mutant (Strain 2, Table 1) in M9 minimal media with or without 1 g/L L-fucose or D-lactose.

FIG. 2B shows expression of P_T7:sfgfp in BL21 Star (DE3) (Strain 3) and ΔlacZ mutant (Strain 4, Table 1) in LB-media. Cultures were induced with or without 1 mM IPTG, respectively. A indicates that the gene was removed from the genome. Error bars indicate s.d. (n=3 biological replicates).

FIG. 3A shows cell growth and protein expression after installation of the T7 RNAP expression system into E. coli K-12-derivative strains. GFP fluorescence assay in K-12 derivative strains, BW25113 Z1 (Strain 7) and MG1655 Z1 (Strain 8, Table 1) with ss9::P_lacuv5:T7rnap. Cultures were induced with or without 1 mM IPTG at 37° C. for 24 h.

FIG. 3B shows a growth assay of MG1655 Z1 (Strain 6) and Strain 10 (Strain 6 with ΔfucU ΔlacZ, Table 1) in M9-minimal media with or without 1 g/L L-fucose or D-lactose at 37° C. for 24 h.

FIG. 3C shows a fluorescence assay to evaluate GFP expression from P_T7in Strain 8 and Strain 12 (Strain 8 with ΔfucU ΔlacZ, Table 1). Cultures were grown in LB-media and induced with or without 1 mM IPTG at 37° C. for 24 h.

FIG. 3D shows a growth assay of MG1655 Z1 and Strain 15 (MG1655 Z1 with ΔfucU ΔlacZ, Table 1) in M9-minimal media with 1 g/L L-fucose or D-lactose. A indicates gene was removed from the genome. Error bars indicate s.d. (n=3 biological replicates).

FIG. 4A, taken with FIG. 4B, shows GFP expression under lac-promoter variants in K-12 derivative strains. Cultures were grown in LB-media and induced with or without 1 mM IPTG at 37° C. for 24 h. GFP expression under promoter P_L1acO1in Strain 17 (MG1655 Z1), Strain 18 (MG1655 Z1 with ΔfucU ΔlacZ) and Strain 19 (Strain 18 with ss9::P_lacUV5:T7rnap, Table 1).

FIG. 4B shows GFP expression under promoter P_lacUV5in Strain 20 (MG1655 Z1), Strain 21 (Strain 20 with ΔfucU) and Strain 22 (Strain 20 with ΔfucU ΔlacZ, Table 1). A indicates gene was removed from the genome. Error bars indicate s.d. (n=3 biological replicates).

FIG. 5 shows the effects of carbon sources on LDFT production. Strain 23 (MG1655 Z1 ΔfucU ΔlacZ with the LDFT production plasmids, Table 1) was grown in M9P with 5 g/L glucose or 10 g/L glycerol at 30° C. for 24 h. Cultures were supplemented with 1 g/L lactose and with or without 1 g/L L-fucose and induced with 1 mM IPTG and 100 ng/mL aTc. L-Fucose concentration (diagonal), lactose concentration (checkered), monofucosides (2′-FL/3-FL) concentration (zigzag) and LDFT concentration (filled) were measured at 24 h. Error bars indicate s.d. (n=3 biological replicates).

FIG. 6 shows the additional expression of lactose and L-fucose permease genes to enhance lactose and L-fucose availabilities. Strain 14 (MG1655 Z1 ΔfucU ΔlacZ, Table 1) was used as a host strain. Strain 23 (Strain 14 with the LDFT production plasmids), Strain 24 (Strain 14 with the LDFT production plasmids with lacY), Strain 25 (Strain 14 with the LDFT production plasmids with fucU), and Strain 26 (Strain 14 with the LDFT production plasmids with lacY and fucU) were grown in M9P with 10 g/L glycerol at 30° C. for 24 h. Cultures were supplemented with 1 g/L lactose and 1 g/L L-fucose and induced with 1 mM IPTG and 100 ng/mL aTc. Growth and production of monofucosides (2′-FL/3-FL, zigzag) and LDFT (filled) were determined at 24 h. + indicates the corresponding gene was expressed from the genome and +++ indicates the corresponding gene was additionally expressed from a plasmid. Error bars indicate s.d. (n=3 biological replicates).

FIG. 7A, taken with FIG. 7B, shows the effects of IPTG and aTc concentrations on LDFT production. Strain 26 (MG1655 Z1 ΔfucU ΔlacZ with the LDFT production plasmids with lacY and fucU) was grown in M9P with 10 g/L glycerol at 30° C. for 24 h. Cultures were supplemented with 1 g/L lactose and 1 g/L fucose and induced with 100 ng/mL aTc and various concentrations of IPTG (0, 25, 50, 100 and 1000 μM).

FIG. 7B shows results for Strain 26, grown as described for FIG. 7A except with 50 M IPTG and various concentrations of aTc (0, 25, 50, 100 ng/mL). OD₆₀₀, L-Fucose concentration (diagonal), lactose concentration (checkered), monofucosides (2′-FL/3-FL) concentration (zigzag) and LDFT concentration (filled) were measured at 24 h. Error bars indicate s.d. (n≥3 biological replicates).

FIG. 8A shows growth (OD₆₀₀) of strain 26 (MG1655 z1 ΔfucU ΔlacZ with the LDFT production plasmids with lacY and fucU) in M9P with 20 g/L glycerol at 30° C. for 12 h. Cultures were supplemented with 1 g/L lactose and 1 g/L L-fucose and induced with 50 M IPTG and 100 ng/mL aTc. Error bars indicate s.d. (n=3 biological replicates).

FIG. 8B shows glycerol concentration (cross), fucose concentration (diamond), lactose concentration (triangle), monofucosides (2′-FL/3-FL) concentration (square) and LDFT concentration (circle) were monitored during growth of Strain 26 as described for FIG. 8A. Error bars indicate s.d. (n=3 biological replicates).

FIG. 9 shows sugar levels in cultures upon delayed expression of Hp3/4 ft in Strain 26. Strain 26 (MG1655 Z1 ΔfucU ΔlacZ with the LDFT production plasmids with lacY and fucU) was grown in M9P with 10 g/L glycerol at 30° C. for 24 h. Cultures were supplemented with 1 g/L lactose and 1 g/L L-fucose and induced with 50 μM IPTG at 0 h. 100 ng/mL aTc was added to cultures at 0, 2, 4, and 6 h. OD₆₀₀, monofucosides (2′-FL/3-FL) concentration (zigzag) and LDFT concentration (filled) were measured at 24 h. Error bars indicate s.d. (n=3 biological replicates).

FIG. 10 shows LDFT production with 2′-FL feeding. The wbgL gene was removed from pAL2029, generating pAL2059 (Table 2). Strain 27 (MG1655 Z1 ΔfucU ΔlacZ harboring pAL1760 (Hp3/4 ft) and pAL2059 (fkp, lacY, and fucU), Table 1) was grown in M9P with 10 g/L glycerol at 30° C. for 24 h. Cultures were supplemented with 1.4 g/L 2′-FL (mole equivalent to 1 g/L lactose) and 0.5 g/L L-fucose and induced with 50 μM IPTG and 100 ng/mL aTc. Glycerol concentration (cross), L-fucose concentration (diamond), 2′-FL concentration (square) and LDFT concentration (circle) were monitored during the experiment. Error bars indicate s.d. (n=3 biological replicates).

FIG. 11 shows LDFT production in Strain 26 with various concentrations of lactose and fucose. Strain 26 (MG1655 Z1 ΔfucU ΔlacZ with the LDFT production plasmids with lacY and fucU) was grown in M9P with 20 g/L glycerol at 30° C. for 24 h. Cultures were supplemented with lactose and L-fucose and induced with 50 μM IPTG and 100 ng/mL aTc. OD₆₀₀, L-fucose concentration (diagonal), lactose concentration (dotted), 2′-FL concentration (wave) and LDFT concentration (filled) were measured at 24 h. Error bars indicate s.d. (n≥3 biological replicates).

FIG. 12 shows fucosyltransferases and other enzymes for production of oligosaccharide products in recombination host cells.

DETAILED DESCRIPTION OF THE INVENTION

Provided herein are methods for producing oligosaccharide products, such as tetrasaccharide lactodifucotetraose (LDFT) and other difucosylated oligosaccharides, in recombinant hosts such as E. coli. The present invention is based, in part, on the pairing of glycosyltransferases with complementary substrate specificities, e.g., pairing of α1-2-fucosyltransferases with high activity towards lactose and α1-3-fucosyltransferases with higher activity towards 2′-fucosyllactose (2′-FL) than lactose. The selectivity of the α1-3-fucosyltransferase provides for minimal production of 3-fucosyllactose (3-FL) as a side product, resulting in the production of difucosylated oligosaccharides such as difucosylated tetrasaccharide lactodifucotetraose (LDFT) in high yield.

The use of bacterial fucosyltransferases with narrow acceptor selectivity can drive the sequential fucosylation of acceptors such as lactose and intermediates such as 2′-fucosyllactose (2′-FL) to produce LDFT and other fucosylated products. Deletion of substrate degradation pathways that decouple cellular growth from product fucosylation can enhance expression of native substrate transporters, and modular induction of the genes in relevant biosynthetic pathways allows for complete conversion of acceptors such as lactose into products such as LDFT with only minor quantities of side products such as 3-fucosyllactose (3-FL). In certain embodiments, for example, 5.1 g/L of LDFT can be produced from 3 g/L lactose and 3 g/L L-fucose in 24 h. The results described herein demonstrate promising applications of microbial biocatalysts for the production of multi-fucosylated HMOs.

LDFT can be synthesized from lactose and L-fucose in a two-step fucosylation process using an α1-2-fucosyltransferase and an α1-3-fucosyltransferase. While monofucosylation of lactose with a single fucosyltransferase for the microbial production of 2′-FL and 3-FL has been studied, the effects of implementing an α1-2-fucosyltransferase and an α1-3-fucosyltransferase together in a cellular system to produce a difucosylated HMO has not been reported. As lactose is a suitable acceptor substrate for both fucosyltransferases, both 2′-FL and 3-FL can be produced as mono-fucosylated products in the first fucosylation step of the system with the presence of both fucosyltransferases. It was shown previously that while an α1-3/4-fucosyltransferase from Helicobacter pylori (Hp3/4FT) can use both non-fucosylated and α1-2-fucosylated galactosyl oligosaccharides as substrates (McArthur et al., 2019; Yu et al., 2017), α1-2-fucosyltransferases from Escherichia coli 0126 (WbgL) (Engels and Elling, 2014; McArthur et al., 2019) and Thermosynechococcus elongates (Zhao et al., 2016) are selective towards lactose and other non-fucosylated galactosyl oligosaccharide acceptor substrates.

An E. coli-based system according to the present disclosure, for example, employs two fucosyltransferases that preferentially fucosylates lactose to form a 2′-FL intermediate that is further fucosylated to produce the target LDFT. Various promoter expression systems were assessed to establish heterologous expression of the desired biosynthetic pathway. LDFT production was decoupled from bacterial growth by removing catabolic pathways of starting substrates and by maintaining cell density with glycerol, an inexpensive carbon source that does not activate carbon catabolite repression of lactose and L-fucose transporters (Kopp et al., 2017; Paulsen et al., 1998). To enhance intracellular availability of substrates, the lactose and L-fucose transporter genes, lacY and fucP, were additionally expressed from plasmids. With additional fine-tuning of the expression levels of individual glycosyltransferase genes, the strain produced 5.1 g/L of LDFT from 3 g/L lactose, achieving 910% of the theoretical maximum yield of LDFT in 24 h.

I. Recombinant Host Cells for Production of Oligosaccharides

Provided herein are recombinant cells for the production of oligosaccharide products. The cells include:

- a polynucleotide encoding a first glycosyltransferase polypeptide having a first substrate selectivity, and
- a polynucleotide encoding a second glycosyltransferase polypeptide having a second substrate selectivity.

Glycosyltransferases and other enzymes suitable for use in the methods described herein include, but are not limited to, those summarized in FIG. 12. In some embodiments, the first glycosyltransferase is a fucosyltransferase having a first substrate selectivity and the second glycosyltransferase is a fucosyltransferase polypeptide having a second substrate selectivity.

A. α1-2-fucosyltransferase

Fucosyltransferases are inverting glycosyltransferases and are classified into eight glycosyltransferase (GT) families in the Carbohydrate-Active enZYmes (CAZy) database: GT10, GT11, GT23, GT3, GT56, GT65, GT68 and GT74 (see, cazy.org; Drula, et al. Nucleic Acids Research, 2022, Vol. 50, D571-D577; and references cited therein).

In some embodiments, the first fucosyltransferase polypeptide is an α1-2-fucosyltransferase polypeptide classified by Enzyme Commission number 2.4.1.69. WbgL, according to SEQ ID NO:1, and other GT11 family fucosyltransferases are thought to be GT-B fold glycosyltransferases containing two separate Rossmann domains (characterized by a six-stranded parallel β-sheet with a 321456 topology) with a connecting linker region and a catalytic site between the domains. See, Engels et al. (Glycobiology 2014, 24(2): 170-178) and Breton et al. (Glycobiology 2006, 16(2): 29R-37R). A high degree of conservation has been observed between protein members of the GT-B family, especially in the nucleotide-binding domain at the C-terminus. A glutamate residue and glycine-rich loops are thought to interact with the ribose and phosphate moieties of the nucleotide. The α1-2-fucosyltransferase may be a GT11 family fucosyltransferase having one or more conserved motifs corresponding to residues 8-16 (motif IV), 158-167 (motif I), 201-207 (motif II), and 234-273 (motif III) of SEQ ID NO:1. In some embodiments, the α1-2-fucosyltransferase includes from one to four amino acid sequences having at least 70% identity (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to motif IV, motif I, motif, II, and/or motif III in SEQ ID NO:1. Highly conserved motif I is likely involved in GDP-fucose binding. Residues corresponding to R¹⁶¹and D¹⁶⁴have been indicated to play roles in donor binding and enzyme activity (see, Li, et al. Biochemistry 2008, 47, 11590-11597). In addition to amino acid sequences corresponding to motifs I, II, III, and/or IV, the α1-2-fucosyltransferase may also include on or more acid sequences having at least 70% identity residues 1-7, 17-157, 168-200, 208-233, and/or 274-297 of SEQ ID NO:1.

Percentage of sequence identity can be determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the sequence (e.g., a peptide of the invention) in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence which does not comprise additions or deletions, for optimal alignment of the two sequences. The percentage can be calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

“Identical” and “identity,” in the context of two or more polypeptide sequences or nucleic acid sequences, refer to two or more sequences or subsequences that are the same. Sequences are “substantially identical” to each other if they have a specified percentage of nucleotides or amino acid residues that are the same (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.

For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analyses is publicly available at the National Center for Biotechnology Information website, ncbi.nlm.nih.gov. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits acts as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=1, N=−2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see, e.g., Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

Examples of α1-2-fucosyltransferases include, but are not limited to, E. coli O126 α1-2-fucosyltransferase (“WbgL;” GenBank: ABE98421.1; SEQ ID NO:1), H. mustelae 12198 α1-2-fucosyltransferase (“Hm2FT;” GenBank: CBG40460; SEQ ID NO:8), E. coli 0128:B12 α1-2-fucosyltransferase (“WbsJ;” GenBank: AA037698.1; SEQ ID NO:9), H. pylori UA1234 α1-2-fucosyltransferase (“Hp2FTa;” GenBank: AAD29863.1; SEQ ID NO:10), and H. pylori UA802 α1-2-fucosyltransferase (“Hp2FTb;” GenBank: AAC99764.1; SEQ ID NO:11). In some embodiments, the α1-2-fucosyltransferase polypeptide comprises an amino acid sequence having at least 70% identity (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to WbgL (SEQ ID NO: 1), Hm2FT (SEQ ID NO:8), WbsJ (SEQ ID NO:9), Hp2FTa (SEQ ID NO:10), or Hp2FTb (SEQ ID NO:11).

In some embodiments, the α1-2-fucosyltransferase polypeptide is an E. coli 0126 α1-2-fucosyltransferase WbgL polypeptide.

B. α1-3-fucosyltransferase

In some embodiments, the second fucosyltransferase polypeptide is an α1-3-fucosyltransferase polypeptide having, for example, β-LacNac α-1,3-L-fucosyltransferase activity (EC 2.4.4.1), galactoside α-1,3/1,4-L-fucosyltransferase activity (EC 2.4.1.65), or galactoside α-1,3-L-fucosyltransferase activity (EC 2.4.1.152). The α1-3-fucosyltransferase may be a GT10 family fucosyltransferase or a GT11 family fucosyltransferase. In some embodiments, the GT10 fucosyltransferase has a glycosyltransferase B (GT-B) fold containing two separated Rossmann domains as described, for example, by Breton et al. supra.

Examples of α1-3-fucosyltransferases include, but are not limited to H. pylori UA948 α1-3/4-fucosyltransferase (“Hp3/4FT;” GenBank: AAF35291.2; SEQ ID NO:3), H. pylori ATCC43504 α1-3-fucosyltransferase (“Hp43504 3FT;” GenBank: AAB93985; SEQ ID NO:12), H. pylori J99 α1-3-fucosyltransferase (“HpJ99 3FT;” GenBank: AAD06169.1, AAD06573.1; SEQ ID NOS:13-14), H. pylori NCTC11637 α1-3-fucosyltransferase (“Hp11637 3FT;” GenBank: AAB93985; SEQ ID NO:15), B. fragilis NCTC 9343 α1-3/α1-4-fucosyltransferase polypeptide (“Bf3/4FT;” GenBank: CAH09495.1; SEQ ID NO:16), and H. hepaticus ATCC 51449 Hh0072 (“Hh0072”; GenBank: AAP76669.1; SEQ ID NO:17). In some embodiments, the α1-2-fucosyltransferase polypeptide comprises an amino acid sequence having at least 70% identity (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to Hp3/4FT (SEQ ID NO:3), Hp43504 3FT (SEQ ID NO:12), HpJ99 3FT (SEQ ID NOS:13 and/or 14), Hp11637 3FT (SEQ ID NO:15), Bf3/4FT (SEQ ID NO:16), or Hh0072 (SEQ ID NO:17).

In some embodiments, the α1-3-fucosyltransferase polypeptide is a truncated α1-3-fucosyltransferase polypeptide, e.g., residues 1-428 of SEQ ID NO:2, or a polypeptide having at least 70% identity to residues 1-428 of SEQ ID NO:2.

In some embodiments, the cells further include one or more polynucleotides selected from the group consisting of:

- a polynucleotide encoding a nucleotide sugar pyrophosphorylase,
- a polynucleotide encoding a monosaccharide transporter such as a fucose transporter, and
- a polynucleotide encoding an oligosaccharide transporter such as a lactose transporter.

C. Kinase/pyrophosphorylases

In some embodiments, the nucleotide sugar pyrophosphorylase polypeptide is a bifunctional glycokinase and nucleotide sugar pyrophosphorylase polypeptide. In some embodiments, the bifunctional enzyme is an L-fucokinase/GDP-fucose pyrophosphorylase (Fkp). Fkps are a class of enzymes that catalyze two steps of the L-fucose salvage pathway for the geeneration of activated GDP-L-fucose via a fucose-1-phosphate intermediate. Fkps have been observed to adopt a tetrameric formation, with each monomer containing an N-terminal GDP-fucose pyrophosphorylase domain, an intermediate linking domain, and a C-terminal fucokinase domain. The pyrophosphorylase domain contains a Rossmann fold and a left-handed β-helix, and the fucokinase contains a GHMP sugar kinase fold. The linker between the two domains contains α-helices. Examples of Fkps include, but are not limited to Bacteroides fragilis bifunctional L fucokinase/GDP-L-fucose pyrophosphorylase (“BfFKP;” GenBank: CAH08307.1; SEQ ID NO:3) and Arabidopsis thaliana bifunctional fucokinase/fucose pyrophosphorylase (“AtFKGP;” UniProt: Q9LNJ9; SEQ ID NO:18).

D. Glycotransporters

In some embodiments, the monosaccharide transporter is a fucose transporter, and the oligosaccharide transporter is a lactose transporter. Many such transporters belong to the major facilitator superfamily (MFS), which shuttle substrates across cell membranes by leveraging electrochemical potential. MFS transporters such as E. coli LacY are composed of 12 transmembrane helices, with the six N-terminal and the six C-terminal helices forming distinct helical bundles connected by a loop. The two bundles have the same topology and exhibit pseudo-two-fold symmetry around an axis perpendicular to the membrane bilayer. A hydrophilic cavity is defined by helices 1, 2, 4, and 5 in the N-terminal bundle and helices 7, 8, 9, and 11 in the C-terminal bundle, while helices 3, 6, 9, and 12 are largely embedded in the membrane. In some embodiments, the lactose transporter polypeptide is an E. coli LacY polypeptide. Similar lactose transporters have been identified in Citrobacter spp., Cronobacter spp., Enterobacter spp., Klebsiella spp., Salmonella spp., and Shigella spp., and may also be incorporated in the recombinant host cells. In some embodiments, the lactose transporter polypeptide comprises an amino acid sequencing having at least 70% identity (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the E. coli str. K-12 substr. MG1655 LacY set forth in SEQ ID NO:4.

In some embodiments, the L-fucose transporter polypeptide is an E. coli FucP polypeptide. Similar fucose transporters have been identified in species including, but not limited to, Chryseobacterium mucoviscidosis, Enterobacter hormaechei, Escherichia albertii, Klebsiella pneumoniae, Salmonella enterica, and Shigella flexneri, and may also be incorporated into the recombinant host cells. In some embodiments, the lactose transporter polypeptide comprises an amino acid sequencing having at least 70% identity (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the E. coli str. K-12 substr. MG1655 FucP set forth in SEQ ID NO:5.

In some embodiments, the cell further includes a polynucleotide encoding an additional transporter polypeptide. In some embodiments, the additional transporter polypeptide is a Bifidobacterium fucosyllactose transporter polypeptide, e.g., those including the domains set forth in SEQ ID NOS:19-21 and/or SEQ ID NOS:22-24.

Suitable microbial hosts include, but are not limited to, members of the genera Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Pichia, Candida, Hansenula and Saccharomyces. Preferred hosts include: Escherichia coli, Alcaligenes eutrophus, Bacillus licheniformis, Paenibacillus macerans, Rhodococcus erythropolis, Pseudomonas putida, Lactobacillus plantarum, Enterococcus faecium, Enterococcus gallinarium, Enterococcus faecalis, Bacillus subtilis and Saccharomyces cerevisiae. In some embodiments, the cell is an E. coli cell, a B. subtilis cell, a C. glutamicum cell, or an S. cerevisiae cell. In some embodiments, the cell is an E. coli BW25113 Z1 cell or an E. coli MG1655 Z1 cell.

Recombinant organisms containing the genes encoding glycosyltransferases and other enzymes for the production of human milk oligosaccharides and other oligosaccharide products can be constructed using techniques well known in the art. Polynucleotide sequences may be obtained from various organisms as described above, e.g., from a bacterial genome. For example, if the sequence of the gene is known, suitable genomic libraries may be created by restriction endonuclease digestion and may be screened with probes complementary to the desired gene sequence. Once the sequence is isolated, the DNA may be amplified using standard primer-directed amplification methods such as polymerase chain reaction to obtain amounts of DNA suitable for transformation using appropriate vectors. Tools for codon optimization for expression in a heterologous host are readily available.

Once the relevant pathway genes are identified and isolated they may be transformed into suitable expression hosts by means well known in the art. Vectors or cassettes useful for the transformation of a variety of host cells are common and commercially available from companies such as Thermo Fisher Scientific (Waltham, MA), MilliporeSigma (La Jolla, CA), and New England Biolabs, Inc. (Burlington, MA). Typically the vector or cassette contains sequences directing transcription and translation of the relevant gene, a selectable marker, and sequences allowing autonomous replication or chromosomal integration. Such vectors may include a region upstream of the gene which harbors transcriptional initiation controls and a region downstream of the gene which controls transcriptional termination. Both control regions may be derived from genes homologous to the transformed host cell, although it is to be understood that such control regions may also be derived from genes that are not native to the specific species chosen as a production host.

Initiation control regions or promoters, which are useful to drive expression of the relevant pathway coding regions in the desired host cell are numerous and familiar to those skilled in the art. Promoters capable of driving these genetic elements include, but are not limited to, CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI, CUP1, FBA, GPD, and GPM (useful for expression in Saccharomyces); AOX1 (useful for expression in Pichia); and lac, ara, tet, trp, IPL, IPR, T7, tac, and trc (useful for expression in Escherichia coli, Alcaligenes, and Pseudomonas); the amy, apr, npr promoters and various phage promoters useful for expression in Bacillus subtilis, Bacillus licheniformis, and Paenibacillus macerans; nisA (useful for expression Gram-positive bacteria, Eichenbaum et al. Appl. Environ. Microbiol. 64(8):2763-2769 (1998)); and the synthetic P11 promoter (useful for expression in Lactobacillus plantarum, Rud et al., Microbiology 152:1011-1019 (2006)). Termination control regions, if present, may also be derived from various genes native to the preferred hosts.

In some embodiments, the cell is transformed with a first expression vector comprising:

- the polynucleotide encoding the first fucosyltransferase polypeptide,
- the polynucleotide encoding the nucleotide sugar pyrophosphorylase polypeptide,
- the polynucleotide encoding the lactose transporter polypeptide, and
- the polynucleotide encoding the L-fucose transporter polypeptide.

In some embodiments, the polynucleotide encoding the first fucosyltransferase polypeptide (e.g., WbgL) and the polynucleotide encoding the nucleotide sugar pyrophosphorylase polypeptide (e.g., Fkp) are operably linked to a first inducible promoter. In some embodiments, the first inducible promoter is a P_L1acO1promoter.

In some embodiments, the polynucleotide encoding the second fucosyltransferase polypeptide (e.g., Hp3/4FT) is operably linked to a second inducible promoter. In some embodiments, the second inducible promoter is a P_LtetO1promoter.

In some embodiments, the polynucleotide encoding the lactose transporter polypeptide and the polynucleotide encoding the L-fucose transporter polypeptide are operably linked to a constitutive promoter.

In some embodiments, the cell is modified to eliminate or reduce expression of an L-fucose mutarotase and/or a β-galactosidase. In some embodiments, the L-fucose mutarotase is an E. coli fucU, as set forth in SEQ ID NO:6, or a polypeptide having at least 70% identity thereto. In some embodiments, the β-galactosidase is an E. coli LacZ; as set forth in SEQ ID NO:6, or a polypeptide having at least 70% identity thereto. Knockout of L-fucose mutarotases and β-galactosidases can be conducted as described in more detail below. Other CRISPR/Cas9-based strategies, e.g., as described by Zhao et al. (Microb Cell Fact 2016, 15: 205) or König et al (Bio Protoc. 2018, 8(2): e2688), may be employed, as well as methods employing phage λ Red recombinase and/or FLP recombinase (see, Datsenko and Wanner. PNAS, 2000, 97 (12): 6640-6645; Baba, et al. Molecular Systems Biology 2006, 2:2006.0008)

II. Production of Oligosaccharides

Also provided herein are methods for producing oligosaccharide products. In some embodiments, the oligosaccharide product includes two or more fucose moieties, and the method comprising culturing a recombinant cell as described herein in a cell culture medium comprising L-fucose, an oligosaccharide acceptor, and a carbon source. The cell is cultured under conditions in which a nucleotide sugar pyrophosphorylase polypeptide, a first fucosyltransferase polypeptide, a second fucosyltransferase polypeptide, a lactose transporter polypeptide, and/or an L-fucose transporter polypeptide are expressed and the oligosaccharide acceptor is converted to a fucosylated oligosaccharide.

In some embodiments, the acceptor is selected from the group consisting of lactose, lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT); lacto-N-hexaose (LNH); lacto-N-neohexaose (LNnH); para-lacto-N-hexaose (pLNH); and para-lacto-N-octaose (pLNO). Oligosaccharide products include, but are not limited to lactodifucotetraose (LDFT), difucosyl lacto-N-tetraose (DF-LNT), trifucosyl lacto-N-tetraose (TriF-LNT), trifucosyl para-lacto-N-hexaose (TriF-pLNH), and trifucosyl para-lacto-N-octaose (Tetra-F-pLNO)

In some embodiments, the oligosaccharide acceptor is lactose and the oligosaccharide product is lactodifucotetraose (LDFT).

Cell culture media generally contain a carbon source. Suitable substrates include, but are not limited to, monosaccharides such as glucose and fructose, oligosaccharides such as lactose or sucrose, polysaccharides such as starch or cellulose or mixtures thereof and unpurified mixtures from renewable feedstocks such as cheese whey permeate, comsteep liquor, sugar beet molasses, and barley malt. Additionally the carbon substrate may also be one-carbon substrates such as carbon dioxide or methanol. In addition to one and two carbon substrates, methylotrophic organisms are also known to utilize a number of other carbon containing compounds such as methylamine, glucosamine, and a variety of amino acids for metabolic activity. For example, methylotrophic yeast are known to utilize the carbon from methylamine to form trehalose or glycerol. In some embodiments, the carbon source comprises glucose, glycerol, or a combination thereof.

In addition to an appropriate carbon source, fermentation media will typically contain suitable minerals, salts, cofactors, buffers and other components, known to those skilled in the art, suitable for the growth of the cultures and promotion of the enzymatic pathway for production of the desired oligosaccharide.

Typically, recombinant host cells are grown at a temperature in the range of about 20° C. to about 40° C. in an appropriate medium such as Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth or Yeast medium (YM) broth. Other defined or synthetic growth media may also be used, and the appropriate medium for growth of the particular microorganism will be known by one skilled in the art of microbiology or fermentation science. The use of agents known to modulate catabolite repression directly or indirectly, e.g., cyclic adenosine 2′:3′-monophosphate, may also be incorporated into the fermentation medium.

Suitable pH ranges for the fermentation are between pH 5.0 to pH 9.0, where pH 6.0 to pH 8.0 is preferred as the initial condition. Oligosaccharide production may be conducted under aerobic or anaerobic conditions, including microaerobic conditions.

In some embodiments, expression of the nucleotide sugar pyrophosphorylase polypeptide and the first fucosyltransferase polypeptide is induced at a level corresponding to 30-40% of maximum level (e.g., with isopropyl β-D-1-thiogalactopyranoside in an amount around 50 μM). In some embodiments, expression of the second fucosyltransferase polypeptide is induced at a maximum level (e.g., with anhydrotetracycline at around 100 ng/mL).

The terms “about” and “around,” as used herein to modify a numerical value, indicate a close range surrounding that explicit value. If “X” were the value, “about X” or “around X” would indicate a value from 0.8 X to 1.2 X, preferably a value from 0.9 X to 1.1 X, and, more preferably, a value from 0.95 X to 1.05 X. Any reference to “about X” or “around X” specifically indicates at least the values X, 0.9 X, 0.91 X, 0.92 X, 0.93 X, 0.94 X, 0.95 X, 0.96 X, 0.97 X, 0.98 X, 0.99 X, 1.01 X, 1.02 X, 1.03 X, 1.04 X, 1.05 X, 1.06 X, 1.07 X, 1.08 X, 1.09 X, and 1.10 X. Thus, “about X” and “around X” are intended to teach and provide written description support for a claim limitation of, e.g., “0.98 X.”

The amount of oligosaccharide produced in the cell culture medium can be determined using a number of methods known in the art, for example, high performance liquid chromatography (HPLC) or thin-layer chromatography (TLC).

Oligosacharides may be produced in a batch fashion or continuous fashion. A classical batch fermentation is a closed system where the composition of the medium is set at the beginning of the fermentation and not subject to artificial alterations during the fermentation. Within batch cultures, cells may moderate through a static lag phase to a high growth log phase and finally to a stationary phase where growth rate is diminished or halted. If untreated, cells in the stationary phase will eventually die. Cells in log phase generally are responsible for the bulk of production of end product or intermediate. A variation on the standard batch system is the fed-batch system. Fed-batch fermentation processes typically include incremental addition of an oligosaccharide acceptor or other substrate as the fermentation progresses.

Continuous fermentation typically involves an open system where a defined fermentation medium is added continuously to a bioreactor and an equal amount of conditioned media is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in log phase growth. Continuous fermentation allows for the modulation of one factor or any number of factors that affect cell growth or end product concentration. For example, a limiting nutrient such as the carbon source may be maintained at a fixed rate while all other parameters may be allowed to vary. In other systems a number of factors affecting growth can be altered continuously while the cell concentration, measured by media turbidity, is kept constant. Batch, fed-batch, and continuous fermentation systems are described, for example, by Bull et al. (Eds.) (Manual of Industrial Microbiology and Biotechnology, Third Edition (2010) ASM Press, Washington DC.) which is incorporated herein by reference.

III. Examples
Methods

Reagents

All enzymes involved in the molecular cloning experiments were purchased from New England Biolabs (NEB). All synthetic oligonucleotides were synthesized by Integrated DNA Technologies. Sanger sequencing was provided by Genewiz. D-Lactose was purchased from Sigma-Aldrich. L-Fucose was purchased from V-Labs, Inc. An analytical standard of 2′-FL was purchased from Carbosynth.

For synthesizing 3-FL, 8 mg lactose, L-fucose (1.3 equiv.), adenosine 5′-triphosphate (ATP, 1.3 equiv.), and guanidine 5′-triphosphate (GTP, 1.3 equiv.) were dissolved in 2.3 mL of 100 mM Tris-HCl buffer (pH 7.5) containing 20 mM MgCl₂, 0.35 mg Bacteroides fragilis bifunctional L-fucokinase/GDP-L-fucose pyrophosphorylase (BfFKP) (Yi et al., 2009), 0.15 mg Pasteurella multocida inorganic pyrophosphatase (PmPpA) (Yu et al., 2010), and 0.3 mg Hp3/4FT. The reaction mixture was incubated at 30° C. at 100 rpm for 16 h. The product formation was monitored by liquid chromatography-mass spectrometry (LCMS) (Shimadzu). When all lactose was converted to 3′-FL, the reaction was stopped by adding an equivalent volume of ice-cold ethanol. The mixture was kept at 4° C. for 30 min then centrifuged at 6,900 g for 30 min. The precipitates were removed and the supernatant was concentrated with a rotary evaporator and then passed through a Dowex® 1×8 ion exchange column. The partially purified product was obtained by elution with water. The eluate was concentrated, passed through a Bio-Gel P-2 gel filtration column, and eluted with water. The fractions containing the pure 3-FT product were collected and lyophilized.

To synthesize the LDFT standard, 8 mg lactose, L-fucose (1.2 equiv.), ATP (1.2 equiv.), and GTP (1.2 equiv.) were dissolved in 2.3 mL of 100 mM Tris-HCl buffer (pH 7.5) containing 20 mM MgCl₂, 0.3 mg BfFKP, 0.1 mg PmPpA, and 0.2 mg Helicobacter mustelae α1-2-fucosyltransferase (Hm2FT) (Ye et al., 2019). The reaction mixture was incubated at 30° C. at 100 rpm for 16 h. The product formation was monitored by LCMS. When all lactose was converted to 2′-FL, the reaction mixture was concentrated and applied to the next fucosylation step without purification. In the second step, the reaction mixture containing 10 mM 2′-FL formed from the previous step, L-fucose (1.2 equiv.), ATP (1.2 equiv.), and GTP (1.2 equiv.) in 2.3 mL of 100 mM Tris-HCl buffer (pH 7.5) containing 20 mM MgCl₂, 0.35 mg BfFKP, 0.15 mg PmPpA, and 0.3 mg Hp3/4FT. The reaction mixture was incubated at 30° C. at 100 rpm for 16 h. When all 2′-FL was converted to LDFT as monitored by LCMS, the reaction was stopped by adding an equal volume of ice-cold ethanol. The mixture was kept at 4° C. for 30 min and then centrifuged at 6,900 g for 30 min. The precipitates were removed and the supernatant was concentrated with a rotary evaporator and then passed through a Dowex® 1×8 ion exchange column. The partially purified product was obtained by elution with water. The eluate was concentrated, passed through a Bio-Gel P-2 gel filtration column, and eluted with water. The fractions containing the pure LDFT product were collected and lyophilized.

Strains and Plasmids

All strains used in this study are listed in Tables 1 and 3. All plasmids and primers are listed on Tables 4 and 5. Gene deletions and integrations were constructed using CRISPR-Cas9-mediated homologous recombination (Jiang et al., 2015). Linear DNA repair fragments for gene deletions were constructed by PCR assembly or amplification from genomic DNA using primers listed in Tables 4 and 6. The linear DNA repair fragment for ss9::P_lacUV5:T7rnap was PCR amplified from repair plasmid pAL1856 constructed from pSS9 template (Addgene plasmid #71655) (Bassalo et al., 2016) listed in Tables 3 & 6. All genomic modifications were PCR and sequence verified.

TABLE 1

Strain list

Strain

no.

E. coli strain
Plasmid
Key Genotype

1
AL3535

As BL21 Star (DE3), but ΔlacZ

2
AL3535
pAL1779/pAL1817
ΔlacZ, P_T7:fkp-wbgL, P_T7:Hp3/4ft

3
BL21 Star
pAL1834
P_T7:sfgfp

(DE3)

4
AL3535
pAL1834
ΔlacZ, P_T7: sfgfp

5
AL3600

As AL62, but ss9::PlacUV5:T7rnap

6
AL3601

As AL1050, but ss9::PlacUV5:T7rnap

7
AL3600
pAL1834
P_T7:sfgfp

8
AL3601
pAL1834
P_T7:sfgfp

9
AL3606

As Strain 6, but ΔfucU

10
AL3659

As Strain 9, but ΔlacZ

11
AL3659
pAL1779/pAL1817
ΔfucU, ΔlacZ, P_T7:fkp-wbgL

12
AL3659
pAL1834
ΔfucU, ΔlacZ, P_T7:sfgfp

13
AL3585

As AL1050, but ΔfucU

14
AL3664

As Strain 13, but ΔlacZ

15
AL3732

As Strain 14, but ss9::P_lacUV5:T7rnap

16
AL3732
pAL1834
ΔfucU, ΔlacZ, ss9::P_lacUV5:T7rnap, P_T7:sfgfp

17
AL1050
pAL421
P_LlacO1:sfgfp

18
AL3664
pAL421
ΔfucU, ΔlacZ, P_LlacO1:sfgfp

19
AL3732
pAL421
ΔfucU, ΔlacZ, ss9::P_lacUV5:T7rnap, P_LlacO1:sfgfp

20
AL1050
pAL2054
P_lacUV5:sfgfp

21
AL3585
pAL2054
ΔfucU, P_lacUV5:sfgfp

22
AL3664
pAL2054
ΔfucU, ΔlacZ, P_lacUV5:sfgfp

23
AL3664
pAL1759/pAL1760
ΔfucU, ΔlacZ, P_LlacO1:fkp-wbgL, P_LtetO1:Hp3/4ft

24
AL3664
pAL2027/pAL1760
ΔfucU, ΔlacZ, P_LlacO1:fkp-wbgL,

BBa_K1824896:lacY, P_LtetO1:Hp3/4ft

25
AL3664
pAL2028/pAL1760
ΔfucU, ΔlacZ, P_LlacO1:fkp-wbgL,

BBa_K1824896:fucP, P_LtetO1:Hp3/4ft

26
AL3664
pAL2029/pAL1760
ΔfucU, ΔlacZ, P_LlacO1:fkp-wbgL,

BBa_K1824896:lacY-fucP, P_LtetO1:Hp3/4ft

27
AL3664
pAL2059/pAL1760
ΔfucU, ΔlacZ, P_LlacO1:fkp,

BBa_K1824896:lacY-fucP, P_LtetO1:Hp3/4ft

TABLE 2

Plasmid list

Plasmid
Genotype
Reference

pAL421
P_LlacO1:sfgfp; ColE1; amp^r
This study

pAL1759
P_LlacO1:fkp-wbgL; ColE1; amp^r
This study

pAL1760
P_LtetO1:Hp3/4ft; ColA; kan^r
This study

pAL1779
P_T7: fkp-wbgL; pBR322; amp^r
This study

pAL1817
P_T7:Hp3/4ft; ColA; kan^r
This study

pAL1834
P_T7:sfgfp; pBR322; amp^r
This study

pAL2027
BBa_K1824896:lacY; P_LlacO1:fkp-wbgL;
This study

pBR322; amp^r

pAL2028
BBa_K1824896:fucP; P_LlacO1:fkp-wbgL
This study

pBR322; amp^r

pAL2029
BBa_K1824896:lacY-fucP; P_LlacO1:fkp-wbgL;
This study

pBR322; amp^r

pAL2054
P_lacUV5:sfgfp; ColE1; amp^r
This study

pAL2059
BBa_K1824896:lacY-fucP; P_LlacO1:fkp; pBR322;
This study

amp^r

Other plasmids information is in Table 4.

TABLE 3

Strains used in this study

Strain
Genotype
Source

XL-1 Blue
recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac
Agilent

[F′ proAB lacIq ZΔM15 Tn10 (tet^r)]

BL21 Star
F⁻ ompT hsdS_B(r_B⁻, m_B⁻) gal dcm rne131 (DE3)
ThermoFisher

(DE3) (AL15)

BW25113 Z1
lacI⁺rrnB_T14ΔlacZ_WJ16hsdR514
This study

(AL62)
ΔaraBAD_AH33ΔrhaBAD_LD78rph-1

Δ(araB-D)567 Δ(rhaD-B)568 ΔlacZ4787(::rrnB-

3) hsdR514 rph-1 attB::lacI^qtetR spec^r

MG1655 Z1
F− lambda- ilvG- rfb-50 rph-1 attB::lacI^qtetR spec^r
(Yoneda et al.

(AL1050)

2014)

AL3271
As BW25113, but F′ [proAB lacIq ZΔM15 Tn10 (tet^r)]
This study

ΔfucU

AL3535
As BL21 Star (DE3), but ΔlacZ
This study

AL3585
As AL1050, but ΔfucU
This study

AL3600
As AL62, but ss9::P_lacUV5:T7rnap
This study

AL3601
As AL1050, but ss9::P_lacUV5:T7rnap
This study

AL3606
As AL3601, but ΔfucU
This study

AL3659
As AL3606, but ΔlacZ
This study

AL3664
As AL3585, but ΔlacZ
This study

AL3732
As AL3664, but ss9::P_lacUV5:T7rnap
This study

TABLE 4

Plasmids used in this study

Plasmid
Genotype
Source

pCas
P_cas:cas9 P_araB:Red lacI^qP_trc:sgRNA
Addgene #62225

pMB1 repA101(Ts) kan^r
(Jiang et al., 2015)

pTargetF
sgRNA-pmB1 pMB1 spec^r
Addgene #62226

(Jiang et al., 2015)

pss9 integration
HR1^#-P_T7A1:gfpUV-HR2{circumflex over ( )} pBR322 tet^r
Addgene #71655

template

(Bassalo et al.,

2016)

pAL421
P_LlacO1:sfgfp ColE1 amp^r
This study

pAL631
P_LlacO1:sfgfp ColE1 kan^r
This study

pAL1023
P_LtetO1ColA kan^r
This study

pAL1354
P_LlacO1ColE1 amp^r
This study

pAL1687
P_T7:fkp pBR322 amp^r
This study

pAL1688
P_T7:wbgL pBR322 amp^r
This study

pAL1689
P_T7:Hp3/4ft pBR322 amp^r
This study

pAL1759
P_LlacO1:fkp-wbgL ColE1 amp^r
This study

pAL1760
P_LtetO1:Hp3/4ft ColA kan^r
This study

pAL1762
sgRNA-ss9 pMB1 spec^r
This study

pAL1779
P_T7:fkp-wbgL pBR322 amp^r
This study

pAL1817
P_T7:Hp3/4ft ColA kan^r
This study

pAL1783
HR1^#-P_T7A1:gfpUV-HR2{circumflex over ( )} pBR322 amp^r
This study

pAL1834
P_T7:sfgfp pBR322 amp^r
This study

pAL1845
ΔlacZ HR1^#-HR2{circumflex over ( )} ColE1 amp^r
This study

pAL1846
sgRNA-lacZ pMB1 spec^r
This study

pAL1851
sgRNA-lacZ pMB1 amp^r
This study

pAL1853
sgRNA-ss9 pMB1 amp^r
This study

pAL1854
P_lacUV5:lacZα-T7rnap ColE1 amp^r
This study

pAL1855
P_lacUV5:T7rnap ColE1 amp^r
This study

pAL1856
HR1-P_lacUV5:T7rnap-HR2 pBR322 amp^r
This study

pAL1864
sgRNA-fucU pMB1 amp^r
This study

pAL2026
BBa_K1824896*, P_LlacO1:fkp-wbgL
This study

colE1 amp^r

pAL2027
BBa_K1824896*:lacY, P_LlacO1;fkp-wbgL
This study

ColE1 amp^r

pAL2028
BBa_K1824896*:fucP, P_LlacO1:fkp-wbgL
This study

ColE1 amp^r

pAL2029
BBa_K1824896*:lacY-fucP, P_LlacO1:fkp-
This study

wbgL ColE1 amp^r

pAL2054
P_lacUV5:sfgfp ColE1 amp^r
This study

pAL2059
BBa_K1824896*:lacY, P_LlacO1:fkp ColE1
This study

amp^r

^#upstream homologous region,

{circumflex over ( )}downstream homologous region,

*iGEM part #: BBa_K1824896

TABLE 5

Oligonucleotides used in this study

SEQ ID

Plasmid(s)
Used for PCR

NO
Name
Sequence 5′→3′
produced
and/or sequencing

27
AZ52
GTCTTGTCGATCAGGATGATC

pAL1817

28
AZ55
CGAGCCCGTATAAACTGAAAGC

pAL1760

29
AZ56
CTAGGTCTAGGGCGGCGGATTTG

pAL1759,

pAL1845,

pAL1854, pAL2054

30
AZ57
CGTAAGATACTGACAGAAAACGC

pAL1759,

pAL1779, pAL2059

31
AZ60
GGAGGAAGGAAAGAATATCTGG

pAL1759

32
AZ61
GTGACTTTATTGGCTGCTATTCC

pAL1759

33
AZ64
CAAATAGGGGTTCCGCGCACAT

pAL1759,

pAL1779,

pAL1854, pAL1855

34
AZ65
GATATGACTGTTCTCGATCCA

pAL1759

35
AZ82
CCCTGGCAAATGTTGATTGA

fucU upstream

36
AZ83
CAGGCTGTTACCAAAGAAGT

fucU downstream

37
AZ105
CGGCCTTATTGTCTCTCTGC

pAL1817

38
AZ154
CCTAGGTCTAGGGCGGCGGATTTG

pAL2059

39
AZ155
CATTATAACATTCTTCAAGCAGCC

pAL2026, pAL2059

40
AZ224
AATTCATTAAAGAGGAGAAAAGATATA
pAL1759

CCATGGGCAGCAG

41
AZ225
CATATGTATATCTCCTTCTTTTATGATCG
pAL1759

TGATACTTGGAATC

42
AZ226
AAGAAGGAGATATACATATGAGCATTA
pAL1759

TTCG

43
AZ227
TTAGCAGCCGGATCTCAGTG
pAL1759

44
AZ228
CACTGAGATCCGGCTGCTAAGGTACCTA
pAL1759

ATCTAGAGGCATC

45
AZ229
TTTCTCCTCTTTAATGAATTCGGTCAGTG
pAL1759

CGTCC

46
AZ230
AATTCATTAAAGAGGAGAAACATATGTT
pAL1760

CCAACCGCTGCTG

47
AZ231
CTCTAGAGTCATTAGGTACCGCTTTGTT
pAL1760

AGCAGCCGGATC

48
AZ233
TTTCTCCTCTTTAATGAATTCGG
pAL1760

49
AZ259
GAATTCGGTCAGTGCGTCCTGCTG

pAL2027, pAL2028

50
AZ274
AAGGATCCGGCTGCTAACAAAAGGAGA
pAL1779

TATACATATGAGC

51
AZ275
ACTCAGCTTCCTTTCGGGCTAGCAGCCG
pAL1779

GATCTCAGTG

52
AZ276
AGCCCGAAAGGAAGCTGAGTTGGCTGC
pAL1779

TG

53
AZ277
TTGTTAGCAGCCGGATCCTTATGATCGT
pAL1779

GATACTTG

54
AZ293
ATGATTGAACAAGATGGATTGCACGC
pAL1817
pAL1817

55
AZ294
AGGAGAGCGTTCACCGACAAAACGCCA
pAL1817

GCAACGCGG

56
AZ295
AATCCATCTTGTTCAATCATACTCTTCCT
pAL1817

TTTTCAATATTATTGAAGCATTTATCAG

GG

57
AZ307
CACTTTACTACCCACGCCGC

BL21 Star (DE3)

attB locus

58
AZ308
GACTGGCAGCAACAGGTGGC

BL21 Star (DE3)

attB locus

59
AZ309
GTTGAGCTACAGGCGGTCAG

ss9::P_lacUV5:T7rnap

60
AZ310
ATTTACTAACTGGAAGAGGC

pAL1854,

pAL1856,

ss9::P_lacuV5:T7rnap

61
AZ311
CATTGAGTCAACCGGAATGG

pAL1854,

pAL1856,

ss9::_PlacuV5:T7rnap

62
AZ312
AAACCAATCGGTAAGGAAGG

pAL1854,

pAL1856,

ss9::P_lacUV5:T7rnap

63
AZ313
TTTTACCGTTCACGCGCTGG

ss9::P_lacUV5:T7rnap

64
AZ336
TGGTGCCGCGCGGCAGCCATATGGGTCA
pAL1834

TCACCACCATCATC

65
AZ337
TTCGGGCTAGCAGCCGGATCTTATTTGT
pAL1834

ACAGTTCGTCCATGCCG

66
AZ338
GATCCGGCTGCTAGCCCGAAAGGAAGC
pAL1834

TGAGTTGGCTG

67
AZ339
ATGGCTGCCGCGCGGCACCAG
pAL1834

68
AZ340
AATGCGCGCCATTACCGAGTCCG
pAL1845
lacZ upstream

69
AZ341
AGCTGTTTCCTGTGTGAAATTGTTATCC
pAL1845

GC

70
AZ342
ATTTCACACAGGAAACAGCTTAATAACC
pAL1845

GGGCAGGCCATGTCTG

71
AZ343
ACTTTCTCAATAAATGCCTCTACTGCTG
pAL1845
lacZ downstream

GCGCACC

72
AZ344
GAGGCATTTATTGAGAAAGTTAATCTAG
pAL1845

AGGCATCAAATAAAACGAAAGGCTCAG

TCG

73
AZ345
ACTCGGTAATGGCGCGCATTGGTCAGTG
pAL1845

CGTCCTGCTGATG

74
AZ347
GCCGACACCAGTTTTAGAGCTAGAAATA
pAL1846

G

75
AZ348
TCCGCCGCCTACTAGTATTATACCTAGG
pAL1846

ACTGAG

76
AZ359
CAGCGGTGGAGTGCAATGTCATGAGTAT
pAL1851

TCAACATTTCCG

77
AZ360
ATCGACTGGCGAGCGGCATCTTACCAAT
pAL1851

GCTTAATCAGTG

78
AZ361
GATGCCGCTCGCCAGTCGATTGGC
pAL1851

79
AZ362
GACATTGCACTCCACCGCTGATGAC
pAL1851

80
AZ364
TCCGGATTTACTAACTGGAAGAGGCACT
pAL1855

AAATG

81
AZ365
AGCTGTTTCCTGTGTGAAATTGTTATCC
pAL1855

GCTC

82
AZ366
CCTTTCGTCTTCACCTCGAGTCACTCATT
pAL1854

AGGCACCCCAGGC

83
AZ367
GGTACCTTAGCAGCCGGATCTTACGCGA
pAL1854

ACGCGAAGTCCGAC

84
AZ368
GATCCGGCTGCTAAGGTACCTAATCTAG
pAL1854

AGGC

85
AZ369
CTCGAGGTGAAGACGAAAGGGCCT
pAL1854

86
AZ370
AGTTGATATGTCAAACAGGTTCACTCAT
pAL1856

TAGGCACCCCAGGC

87
AZ371
CGGCGCTCAGTTGGAATTCAACAACAGA
pAL1856

TAAAACGAAAGGCC

88
AZ372
TGAATTCCAACTGAGCGCCGGTC
pAL1856

89
AZ373
ACCTGTTTGACATATCAACTGCGCC
pAL1856

90
AZ384
GTGATGATGGGTTTTAGAGCTAGAAATA
pAL1864

GC

91
AZ385
CAGCGGCGGTACTAGTATTATACCTAGG
pAL1864

AC

92
AZ403
ACTCTTCCTTTTTCAATATTATTGAAGCA

pAL2027,

TTTATCAGGG

pAL2028,

pAL2029,

pAL2054, pAL2059

93
AZ411
CGCGCGGCACACTAGTATTATACCTAGG

pAL2029

AC

94
AZ710
GTGCCACCTGACGTCTAAGACTAGTACT
pAL2026
pAL2026

CTAGTATTTCTCCTCTTTA

95
AZ711
GCTACTAGAGTACTAGAGTACTAGAGAT
pAL2026

TAAAGAGGAGAAATACTAGAGTACTAG

TCTTA

96
AZ712
TCTCTAGTACTCTAGTACTCTAGTAGCT
pAL2026

AGCACTGTACCTAGGACTGAGCTAGCCG

T

97
AZ713
ACGCCTATTTTTATAGGTTAATGTCATG
pAL2026

ATAATAATGGTTTTGACGGCTAGCTCAG

TCC

98
AZ714
TGACATTAACCTATAAAAATAGGCGTAT
pAL2026

CACGAGGCCCTTTCGTCTTCACCTCGAG

AAT

99
AZ715
TTGTTATCCGCTCACAATGTCAATTGTTA
pAL2026
pAL2026

TCCGCTCACAATTCTCGAGGTGAAGACG

AA

100
AZ716
CAATTGACATTGTGAGCGGATAACAAG
pAL2026

101
AZ717
TCTTAGACGTCAGGTGGCACTTTTCG
pAL2026,

pAL2027,

pAL2028.

pAL2029

102
AZ718
GTGCCACCTGACGTCTAAGATTAAGCGA
pAL2027

CTTCATTCACCTG

103
AZ719
GAGAAATACTAGAGTACTAGATGTACTA
pAL2027

TTTAAAAAACACAAACTTTTGGATG

104
AZ720
CTAGTACTCTAGTATTTCTCCTCTTTAAT
pAL2027,

CTCTAGTAC
pAL2028

105
AZ721
GTGCCACCTGACGTCTAAGATCAGTTAG
pAL2028,

TTGCCGTTTGAGAAC
pAL2029

106
AZ722
GAGAAATACTAGAGTACTAGATGGGAA
pAL2028

ACACATCAATACAAACGCAGAG

107
AZ723
GAAAGAGGGGACAAACTAGTATGGGAA
pAL2029

ACACATCAATACAAACG

108
AZ724
TTGTCCCCTCTTTCTCTAGATTAAGCGAC
pAL2029

TTCATTCACCTGACG

109
AZ819
CTAACTGGAAGAGGCACTAAATGGGTC
pAL2054

ATCACCACCATCATCACG

110
AZ820
GGTACCTTAGCAGCCGGATCTTATTTGT
pAL2054

ACAGTTCGTCCATGCCG

111
AZ821
GATCCGGCTGCTAAGGTACCTAATC
pAL2054

112
AZ822
TTAGTGCCTCTTCCAGTTAGTAAATCCG
pAL2054

G

113
AZ851
CTGCTAAGGTACCTAATCTAGAGGCATC
pAL2059

114
AZ852
CCGGATCTTATGATCGTGATACTTGGAA
pAL2059

TC

115
JO232
GGTTCCGCGCACATTTCCC

pAL1845

116
MMM40
GAGTCAGTGAGCGAGGAAGC

pAL1846,

pAL1851, pAL1864

117
MMM131
GCTTGGTTGAGAATACGCCG

pAL1856, ss9

upstream

118
MMM132
GCCTACGATTACGCATGGCTTG

pAL1856, ss9

downstream

119
SD62
GGCCCTTTCGTCTTCACCTCGAG

pAL1760

120
SL005
AACGCAGTCAGGCACCGTGTATGAGTAT
pAL1783
pAL1783

TCAACATTTCCG

121
SL006
GAGGTGCCGCCGGCTTCCATTTACCAAT
pAL1783
pAL1783

GCTTAATCAGTG

122
SL007
ATGGAAGCCGGCGGCACCTC
pAL1783

123
SL008
ACACGGTGCCTGACTGCGTTAGC
pAL1783

124
YT167
TAATGACTCTAGAGGCATCAAATAA
pAL1760

125
YT054
TTGTCGGTGAACGCTCTCCTG
pAL1817
pAL1817

126
YT400
ATGGGTCATCACCACCATCATCA

pAL1834

127
YT430
CCAGTAGTAGGTTGAGGCCGTTGAG

pAL1834

128
YT092
CTACTCAGGAGAGCGTTCAC

pAL1760

129
YT101
GCTTCCCAACCTTACCAGAG

pAL1760

130
YTC427
CAAGCAGCAGATTACGCGCAG

pAL1851

TABLE 6

Guide for CRISPR-Cas9-mediate gene deletions and insertions

pTargetF
PCR Linear Repair Fragment

Modification
Plasmid
20 bp sgRNA sequence 5′→3′
Primers
Template

ΔfucU
pAL1864
ACCGCCGCTGGTGATGATGG
AZ82 (F), AZ83
AL3271

(SEQ ID NO: 131)
(R)
gDNA

ΔlacZ
pAL1851
AGGCGGCGGAGCCGACACCA
AZ340 (F),
pAL1845

(SEQ ID NO: 132)
AZ343 (R)

ss9::P_lacUV5:T7rnap
pAL1853
TCTGGCGCAGTTGATATGTA
MMM131 (F),
pAL1856

(SEQ ID NO: 133)
MMM132 (R)

Plasmids for sfGFP fluorescence assays, LDFT production, and 3-FL production were constructed using sequence and ligation independent cloning (SLIC) (Li and Elledge, 2007). Plasmids encoding sgRNAs for CRISPR-Cas9-mediated homologous recombination were constructed with Q5 site-directed mutagenesis using a modified template pTargetF (Addgene plasmid #62226). Templates used for DNA amplification and cloning are listed in Table 7. All plasmids were verified by PCR and Sanger sequencing. Culture conditions

Overnight cultures were grown at 37° C., 250 rpm, in 3 mL of Luria-Bertani (LB) media with appropriate antibiotics. Antibiotic concentrations were as follows: spectinomycin (50 μg/mL), ampicillin (200 μg/mL), and kanamycin (50 μg/mL). Growth assays were carried out in M9 minimal medium (33.7 mM Na₂HPO4, 22 mM KH₂PO4, 8.6 mM NaCl, 9.4 mM NH4Cl, 1 mM MgSO₄, 0.1 mM CaCl₂)) including 1000×A5 trace metal mix (2.86 g H₃BO₃, 1.81 g MnCl₂·4H2O, 0.079 g CuSO₄·5H2O, 49.4 mg Co(NO₃)₂·6H2O per liter water). LDFT production was carried out in M9 minimal medium supplemented with 5 g/L yeast extract (M9P). Optical densities were measured at 600 nm (OD₆₀₀) with a Synergy H1 hybrid plate reader (BioTek Instruments, Inc.).

Growth Assays

Overnight cultures were inoculated at 1% in 3 mL of M9 minimal medium supplemented with 1 g/L D-lactose or 1 g/L L-fucose. Cultures were grown at 37° C., 250 rpm, for 24 h and OD₆₀₀was measured.

Fluorescence Assays

Overnight cultures were inoculated at 1% in 3 mL of LB media and grown at 37° C., 250 rpm, until OD₆₀₀reached 0.4-0.6. Cultures were respectively induced with isopropyl β-D-1-thiogalactopyranoside (IPTG, 1.0 mM) and grown at 37° C., 250 rpm, for 24 h.

TABLE 7

Plasmid construction guide

PCR for Vector
PCR for Insert(s)

Primer
Primer

Sequence of

Plasmid
(F)
(R)
Template
Primer (F)
Primer (R)
Template
Interest

pAL1759
AZ228
AZ229
pAL1354
AZ224
AZ225
pAL1687
fkp

AZ226
AZ227
pAL1688
wbgL

pAL1760
YT167
AZ233
pAL1023
AZ230
AZ231
pAL1689
Hp3/4ft

pAL1779
AZ276
AZ277
pAL1687
AZ274
AZ275
pAL1688
wbgL

pAL1817
AZ294
AZ295
pAL1689
AZ293
YT054
pAL1023
ColA-kan^r

pAL1762*
MMM139
MMM140
pTargetF

pAL1783
SL007
SL008
pss9
SL005
SL006
pAL1354
amp^r

pAL1845
AZ344
AZ345
pAL1354
AZ340
AZ341
AL1050 gDNA
400 bp upstream

HR1 lacZ

AZ342
AZ343
AL1050 gDNA
400 bp

downstream HR2

lacZ

pAL1854
AZ368
AZ369
pAL1759
AZ366
AZ367
BL21 Star (DE3)
P_lacUV5:lacZ□

gDNA
T7rnap

pAL1855*
AZ364
AZ365
pAL1854

pAL1856
AZ372
AZ373
pAL1783
AZ370
AZ371
pAL1855
P_lacUV5:T7rnap

pAL1846*
AZ347
AZ348
pTargetT

pAL1851
AZ361
AZ362
pAL1846
AZ359
AZ360
pAL1687
amp^r

pAL1853
AZ361
AZ362
pAL1762
AZ359
AZ360
pAL1687
amp^r

pAL1864*
AZ384
AZ385
pAL1851

pAL1834
AZ338
AZ339
pAL1687
AZ336
AZ337
pAL421
sfgfp

pAL2026
AZ716
AZ717
pAL1759
AZ710, AZ712,
AZ711, AZ713,
N/A
BBa_K1824896

AZ714
AZ715

pAL2027
AZ720
AZ717
pAL2026
AZ718
AZ719
AL1050 gDNA
lacY

pAL2028
AZ721
AZ722
pAL2026
AZ718
AZ719
AL1050 gDNA
fucP

pAL2029
AZ724
AZ717
pAL2027
AZ721
AZ723
AL1050 gDNA
fucP

pAL2054
AZ821
AZ822
pAL1855
AZ819
AZ820
pAL631
sfgfp

pAL2059*
AZ851
AZ852
pAL2029

*Q5-site directed mutagenesis (NEB).

Fluorescence emission was measured at 510 nm with a Synergy H1 hybrid plate reader (BioTek Instruments, Inc.).

LDFT Production

Overnight cultures were inoculated at 1% in 3 mL of M9P supplemented with 5 g/L glucose, 10 g/L glycerol, or 20 g/L glycerol. Cultures were grown at 37° C., 250 rpm, until OD₆₀₀reached 0.4-0.6. Appropriate concentrations of lactose, L-fucose, IPTG, and anhydrotetracycline (aTc) were added and the cultures were grown at 30° C., 250 rpm, for 24 h. The produced LDFT was confirmed by high resolution electrospray ionization mass spectrometry using a Thermo Electron LTQ-Orbitrap Hybrid MS at the Mass Spectrometry Facility in the University of California, Davis.

HPLC Analysis

To measure glycerol, L-fucose, lactose, 2′-FL, 3-FL, and LDFT, cell culture supernatant was analyzed using HPLC (Shimadzu) equipped with a refractive index detector (RID) 10 A and a Luna Omega HILIC Sugar column (Phenomenex). The mobile phase consisted of 100% 70:30 HPLC-grade acetonitrile:MilliQ water was run at a flow rate of 1.0 mL/min for 12 min, with the column oven at 35° C. and RID cell temperature at 40° C.

To prepare samples for HPLC analysis, 125 μL of culture was collected and spun down at 17,000 g for 5 min. 15 μL of culture supernatant or compound standard in water was diluted with 45 μL of MilliQ water and 180 μL of acetonitrile. The mixture was vortexed and spun down at 17,000 g for 5 min. 40 μL of each sample was injected into the column for analysis.

Results

Pathway Design for LDFT Production in E. coli

HMO production does not naturally occur in E. coli, therefore the following three enzymes were employed for the production of LDFT: a bifunctional L-fucokinase/GDP-L-fucose pyrophosphorylase (Fkp) from Bacteroides fragilis (Yi et al., 2009), an α1-2-fucosyltransferase (WbgL) from E. coli 0126 (Engels and Elling, 2014; McArthur et al., 2019), and α1-3/4-fucosyltransferase (Hp3/4FT) from Helicobacter pylori UA948 (Rasko et al., 2000; Yu et al., 2017). Acceptor substrate specificity studies of both WbgL and Hp3/4FT have been reported (Engels and Elling, 2014; Ma et al., 2006; McArthur et al., 2019; Yu et al., 2017). WbgL exhibits high activity towards non-fucosylated acceptor substrates, such as lactose, N-acetyllactosamine (LacNAc), and lactulose, and no activity towards 3-FL. Hp3/4FT has been shown to be highly active towards LacNAc and 2′-fucosyl-LacNAc with low activity towards lactose. The acceptor preferences of the fucosyltransferases allow sequential fucosylation of lactose for the formation of LDFT in the presence of both fucosyltransferases. Fkp uses one ATP and GTP to convert L-fucose to GDP-fucose, which is taken as a donor substrate by WbgL to fucosylate lactose at the C2′ position, forming the intermediate 2′-FL (FIG. 1). Due to its structural similarity to 2′-fucosyl-LacNAc, 2′-FL was hypothesized to be a suitable acceptor substrate for fucosylation by Hp3/4FT to produce LDFT, which is expected to be secreted to the supernatant by native membrane exporter SetA (Liu et al., 1999).

LDFT Production in E. coli B Strains

The relatively low soluble expression level of recombinant fucosyltransferases was of initial concern as a potential cause of bottlenecks for synthesizing fucosylated HMOs in microbial hosts (Nidetzky et al., 2018). In this study, the C-terminal 34-amino acid hydrophobic sequence of Hp3/4FT was truncated to increase its solubility (Yu et al., 2017). To increase the expression of fucosyltransferases, E. coli B strain BL21 Star (DE3) was selected as an LDFT production host. BL21 Star (DE3) is widely used for recombinant protein expression and is capable of high expression via the two-step IPTG-inducible T7 bacteriophage promoter (Rosano and Ceccarelli, 2014). The fkp and wbgL genes were cloned together into an expression vector under a T7-promoter (P_T7, pAL1779, Table 2) and the truncated Hp3/4 ft gene was cloned into a second expression vector under P_T7(pAL1817, Table 2).

Lactose and L-fucose were used as starting substrates for LDFT production, but E. coli is known to catabolize these two sugars for growth. It was hypothesized that minimizing assimilation of L-fucose and lactose for cellular growth would contribute to maximization of LDFT production. Therefore, the strain's ability to grow on these two carbon sources was evaluated to determine which carbon assimilating pathways to remove. Although the BL21 Star (DE3) encodes all genes involved in L-fucose degradation, the strain was not able to grow on L-fucose as the sole carbon source (FIG. 2A). The strain was able to grow on lactose as the sole carbon source (FIG. 2A). When the lacZ gene encoding for a β-galactosidase was deleted in the strain (Table 1: Strain 1), lactose did not enable growth anymore (FIG. 2).

The two plasmids containing the LDFT production pathway (pAL1779 and pAL1817, Tables 2 & 4) were introduced into Strain 1 to form Strain 2 (Table 1). To determine the best carbon source for growth and production, Strain 2 was grown in parallel with glucose, a common feedstock known for its catabolite repression towards lactose importation (Bruckner and Titgemeyer, 2002), and glycerol, an inexpensive feedstock that does not cause catabolite repression. Under both of these culturing conditions, Strain 2 did not produce LDFT nor its precursor, 2′-FL. To examine the expression from P_T7, the plasmid containing sfgfp under P_T7(pAL1843, Table 2) was introduced into BL21 Star (DE3) and Strain 1 to form Strains 3 and 4, respectively (Table 1). Strain 3 produced a strong fluorescent signal after IPTG induction while Strain 4 did not produce fluorescence signal in either induction conditions, suggesting that T7 RNA polymerase expression was lacking (FIG. 2B). Sequencing of the attB integration locus in Strain 1 revealed an excision of the λDE3 lysogen containing P_lacUV5:lacZα-T7rnap. Several attempts were made to remove lacZ from BL21 Star (DE3) without off-target modifications to the λDE3 lysogen but resulted in failure.

Introduction of the T7 RNAP Gene into K-12 Derivative Strains

Due to difficulties in genetically modifying BL21 Star (DE3), P_lacUV5:T7rnap was integrated into the E. coli K-12 derivative strains, BW25113 Z1 and MG1655 Z1 (Table 3). The Z1 fragment containing laci^q, tetR, and spec^rwas integrated into the attB site of these strains. It has been shown that many regions in the E. coli genome are stable and high-efficiency integration sites for heterologous genes (Bassalo et al., 2016), therefore intergenic locus ss9 was chosen as the insertion site for P_lacUV5:T7rnap. The P_lacUV5:T7rnap cassette was integrated into ss9 of BW25113 Z1 and MG1655 Z1 to form Strains 5 and 6, respectively (Table 1).

pAL1834 containing P_T7:sfgfp was introduced into Strains 5 and 6 to form Strains 7 and 8, respectively (Table 1) to assess the repression and induction efficiencies of P_T7through a fluorescence assay. Tight repression of GFP expression without IPTG was observed in Strains 7 and 8 (FIG. 3A). IPTG induction in Strains 7 and 8 increased GFP fluorescence 95-fold and 440-fold, respectively (FIG. 3A). Strain 6 was chosen as the base strain for further genetic modification due to its tighter repression and stronger inducibility of P_T7. The growth of Strain 6 on L-fucose and lactose was tested. Strain 6 was able to grow on L-fucose or lactose as a sole carbon source (FIG. 3B). To remove L-fucose and lactose assimilation, fucU encoding an L-fucose mutarotase and lacZ were deleted to form Strain 10 (Table 1). Strain 10 was not able to grow on L-fucose or lactose as a sole carbon source (FIG. 3B).

The LDFT production plasmids (pAL1779 and pAL1817, Table 2) were introduced into Strain 10 to form Strain 11 (Table 1). Strain 11 was grown to test LDFT production from lactose and L-fucose. Glucose or glycerol was used to maintain cellular growth. Under both conditions, LDFT was not produced in Strain 11. This prompted the examination of the T7 RNA polymerase expression system in Strain 10. pAL1834 containing P_T7:sfgfp was introduced into Strain 10 to form Strain 12 (Table 1). Strain 12 produced strong GFP fluorescence without IPTG induction, indicating the expression from P_T7was leaky in Strain 12 (FIG. 3C). In Strain 10, a mutation in the promoter region of the P_lacUV5:T7rnap cassette was found. The deletion of lacZ in Strain 9 without incurring P_lacUV5mutations was attempted several times, but the attempts were unsuccessful. Without wishing to be bound by any particular theory, it is believed that that the mutations in P_lacUV5are correlated with the CRISPR-Cas9-mediated gene removal of lacZ due to the similarity of the lacZ promoter to P_lacUV5.

To avoid the potential sequence similarity issues observed for P_lacUV5and the native lacZ promoter, the three modifications into MG1655 Z1 were introduced in a different order. First, fucU and lacZ in MG1655 Z1 were deleted to form Strain 13 (ΔfucU) and Strain 14 (ΔfucU ΔlacZ)). Then, P_lacUV5: T7rnap was integrated into the ss9 locus to form Strain 15 (Table 1). Strain 15 was unable to grow on L-fucose or lactose as a sole carbon source (FIG. 3D). Although the P_lacUV5:T7rnap cassette in Strain 15 had no mutations, Strain 15 with pAL1834 harboring P_T7:sfgfp (Table 1: Strain 16) showed leaky GFP expression without IPTG. To determine if other lac-based promoters are deregulated by the strain modifications, pAL421 containing P_LlacO1:sfgfp was introduced into MG1655 z1, Strains 14 and 15 to form Strains 17, 18, and 19, respectively (Table 1) to assess the regulation of the lac-based promoter in these strains. The expressions from P_LlacO1without IPTG were well repressed in Strains 17, 18 and 19 (FIG. 4A). Next, pAL2045 containing P_lacUV5:sfgfp was introduced into MG1655 z1, Strains 13 and 14 to form Strains 20, 21, and 22, respectively (Table 1). The expression of sfgfp in Strains 21 and 22 was leakier than that in Strain 20 (FIG. 4B), suggesting that the deletion of fucU caused the leaky expression of P_lacUV5.

Production of LDFT in K-12 Derivative Strains

Rather than pursuing alternative promoters for T7rnap, other induction systems for the LDFT biosynthetic pathway genes were used. The fkp and wbgL genes were cloned under P_LlacO1(pAL1759, Tables 2 & 4) and the Hp3/4 ft gene was cloned under an aTc-inducible promoter P_LetO1(pAL1760, Tables 2 & 4) (Lutz and Bujard, 1997). The LDFT production plasmids (pAL1759 and pAL1760) were introduced to Strain 14 to form Strain 23 (Table 1). Strain 23 was grown in M9P containing L-fucose and lactose with glucose or glycerol. After 24 h, Strain 23 produced 0.08 g/L 2′-FL and 0.16 g/L LDFT under the glycerol conditions, but neither were produced under the glucose conditions (FIG. 5).

Enhancing Substrate Levels by Overexpressing Transporter Genes

Intracellular availability of L-fucose and lactose is important for efficient LDFT production. It was hypothesized that additional expression of the substrate transporter genes would increase the substrate supply and improve LDFT production. The lactose and L-fucose membrane symporter genes, lacY and fucP, were expressed under a constitutive promoter (iGEM part No. BBa_K1824896, Tables 2 & 4). The lacY gene was expressed from the fkp-wbgL plasmid pAL2027 (Tables 2 & 4). The LDFT production plasmids with lacY (pAL2027 and pAL1760) were introduced into Strain 14 to form Strain 24 (Table 1) but the overexpression of lacY did not improve LDFT production (FIG. 6). The fucP gene was expressed from the fkp-wbgL plasmid pAL2028 (Table 2). The LDFT production plasmids with fucP (pAL2028 and pAL1760) were introduced into Strain 14 to form Strain 25 (Table 1). After 24 h, Strain 25 produced 0.9 g/L LDFT, a 6.9-fold improvement compared to Strain 23.

Next, both lacY and fucU were expressed from the fkp-wbgL plasmid pAL2029 (Table 2). The LDFT-production plasmids with lacY and fucU (pAL2029 and pAL1760) were introduced into Strain 14 to form Strain 26 (Table 1). Strain 26 produced 1.1 g/L LDFT after 24 h, representing 59% of the theoretical maximum yield (TMY) from lactose and accumulated 0.17 g/L 2′-FL and/or 3-FL (FIG. 6). As the HPLC and the MS methods used were unable to discriminate between the two mono-fucosylated lactose, the combined concentrations of 2′-FL and 3-FL are reported here.

Tuning of the Expression Levels of the LDFT Biosynthetic Pathway Genes

To fine-tune the nucleotide activation of L-fucose and the fucosylation reactions, a range of IPTG concentrations (0, 25, 50, 100, and 1,000 μM) were screened for the expression of P_LlacO1:fkp-wbgL in the presence of 100 ng/mL aTc for induction of P_LtetO1:Hp3/4 ft. The best growth, greatest lactose and L-fucose consumption, and the highest level LDFT production (1.6 g/L, 89% of TMY) was observed with 50 μM IPTG (FIG. 7A). A range of aTc concentrations (0, 25, 50 and 100 ng/mL) were tested for the LDFT production in the presence of 50 μM IPTG to determine if adjusting Hp3/4FT expression levels could improve LDFT production. Strain 26 produced more LDFT with higher concentrations of aTc (FIG. 7B). Thus, the induction condition with 50 μM IPTG and 100 ng/mL aTc was used for further studies.

Characterization of LDFT Production

The LDFT production profile in Strain 26 was characterized for 12 h post-induction by monitoring substrate, intermediate, side product, and LDFT levels using HPLC (FIG. 8). LDFT was first detected at 5 h, and between 5 to 10 h the production rate was 0.24 g/L/h (FIG. 8). Monofucosides (2′-FL/3-FL) were accumulated up to 0.3 g/L until lactose was depleted at 8 h and remained constant at ˜0.3 g/L between 8 to 12 h. The lack of monofucoside consumption after 8 h indicated that most of the remaining monofucoside was 3-FL, which was the side product produced by Hp3/4FT from lactose that cannot be fucosylated further by WbgL to produce LDFT.

When WbgL and Hp3/4FT are expressed at the same time, both enzymes can compete to fucosylate lactose into 2′-FL and 3-FL, respectively. In the presence of lactose and 2′-FL, Hp3/4FT can also convert the respective acceptor substrates into 3-FL and LDFT. It was hypothesized that the delayed induction of Hp3/4 ft would decrease the competition between WbgL and Hp3/4FT for lactose and decrease the production of the side product, 3-FL. Therefore, delaying of the Hp3/4FT expression was tested by adding 100 ng/mL aTc at 2, 4, and 6 h. However, the delayed expressions of Hp3/4 ft resulted in increased monofucoside accumulation and decreased LDFT production (FIG. 9). This increase in monofucoside in the supernatant suggests that 2′-FL formed by WbgL may be secreted to media and its reimport may be limited, which decreases the substrate availability of Hp3/4FT for LDFT production.

To examine the import efficiency of 2′-FL, 2′-FL was fed to the production cultures. The wbgL gene was removed from pAL2029 to form pAL2059 (Table 2). pAL2059 and pAL1760 were introduced into Strain 14 to form Strain 27 (Table 1). Strain 27 was grown in M9P with 10 g/L glycerol. Cultures were induced with 50 μM IPTG and 100 ng/mL aTc and supplemented with 1.42 g/L of 2′-FL (mole equivalent to 1 g/L lactose) and 0.5 g/L L-fucose. Lactose was not fed to the cultures and wbgL was not present in system, making it unlikely for Strain 27 to produce 2′-FL and 3-FL. Under these conditions, LDFT should be produced only from the fed 2′-FL. Strain 27 produced only 0.4 g/L LDFT in 24 h, further supporting that the import of 2′-FL is not efficient in E. coli (FIG. 10).

LDFT Production with Higher Substrate Concentrations

Strain 26 consumed 1 g/L lactose within 8 h and LDFT production reached completion at 12 h post-induction (FIG. 8). To evaluate LDFT production with higher substrate concentrations, Strain 26 was grown in M9P with 20 g/L glycerol and various amounts of lactose and L-fucose (1, 2, or 3 g/L) for 24 h. In conditions with only lactose or L-fucose as the added substrate, Strain 26 did not produce any detectable amounts of fucosides. In the presence of both substrates, the increase in LDFT yield was nearly proportional to the increase of substrate concentrations (FIG. 11). Strain 26 consumed 3.0 g/L lactose and 2.6 g/L L-fucose and produced 5.1 g/L LDFT in 24 h. LDFT was produced at 910% of TMY.

Discussion

LDFT has been identified as an effective gastrointestinal and immunological modulator and has the potential to be developed to treat human diseases. Its high cost and limited commercial access make LDFT a desirable target for production in microbial hosts. Systems developed in E. coli, B. subtilis, and S. cerevisiae have successfully produced HMOs such as 2′-FL, 3-FL, LNT, and LNnT, which represent only a small fraction of over 200 naturally occurring HMOs. Developing microbial production systems dedicated to synthesizing HMOs with a higher structural complexity is still challenging. In this study, a microbial system that specifically and efficiently produces LDFT was established.

The greatest challenge of this study was pairing an α1-2-fucosyltransferase with an α1-3-fucosyltransferase that can efficiently produce LDFT with minimal accumulation of monofucoside intermediates. WbgL was chosen to drive lactose fucosylation into 2′-FL because it expresses well in E. coli and has been characterized to prefer β1-4-linked galactose substrates, such as lactose and LacNAc (Engels and Elling, 2014). From acceptor substrate screenings of α1-3-fucosylatransferases, Hp3/4FT was annotated with high activity towards 2′-fucosyl LacNAc, which suggested 2′-FL may also be a suitable acceptor for Hp3/4FT (Ma et al., 2006; Yu et al., 2017). Characterization of LDFT production as described herein demonstrated that Hp3/4FT had preferential activity towards 2′-FL over lactose and LDFT was formed as the dominant product (FIG. 8). The presence of residual monofucosides indicates possible formation of the side product 3-FL, which is an unsuitable acceptor for WbgL (Engels and Elling, 2014). Monofucoside titers were relatively low and can be separated from LDFT in downstream purification processes. Other fucosyltransferases that may be employed include, but are not limited to, α1-2-fucosyltransferases such as Hm2FT (GenBank: CBG40460), E. coli 0128:B12 α1-2-fucosyltransferase WbsJ (GenBank: AA037698.1), H. pylori UA1234 α1-2-fucosyltransferase (Hp2FTa) (GenBank: AAD29863.1), H. pylori UA802 α1-2-fucosyltransferase (Hp2FTb) (GenBank: AAC99764.1), and related eukaryotic α1-2-fucosyltransferases (see, e.g., cazy.org/GT11_characterized.html); as well as α1-3-fucosyltransferases such as H. pylori ATCC43504 α1-3-fucosyltransferase (Hp3FT) (GenBank: AAB93985), H. pylori J99 α1-3-fucosyltransferase (Hp3FT) (GenBank: AAD06169.1, AAD06573.1), H. pylori NCTC11639 α1-3-fucosyltransferase (Hp3FT) (GenBank: AAB93985), and related eukaryotic α1-3-fucosyltransferases and α1-3/4-fucosyltransferases (see, e.g., cazy.org/GT10_characterized.html). It is possible to screen α1-3-fucosyltransferases for lower activity towards lactose and higher activity towards 2′-FL, and also pursue protein engineering strategies to expand α1-2-fucosyltransferase's acceptor substrate range to 3-FL so that this side product can be fucosylated into LDFT.

The rate of LDFT formation was dictated by carbon catabolite repression (CCR) and the activity of sugar transporters, which firmly control the import of carbohydrates across the inner membrane (Görke and Stülke, 2008). It has been shown that import of glucose through the phosphotransferase system inhibits transcription of lac operon genes, including lacY. From the experiments described above, glucose conditions led to suppressed LDFT production while glycerol conditions resulted in improved LDFT production. This suggests glucose inhibits lactose import whereas glycerol allows for lactose import through sufficient lacY expression. Although glucose is a traditional carbon feedstock for microbial fermentation, it is unsuitable for HMO production systems that use lactose as a substrate. In the absence of CCR, LDFT production was still limited by the native expression levels of lacY and fucP (FIG. 6). Additional expression of fucP increased LDFT production by 6.9-fold to 0.9 g/L (FIG. 6), indicating that L-fucose import was one of the bottlenecks for LDFT production. While native expression levels of lacY without CCR were adequate for supplying lactose, overexpression of lacY and fucU further balanced the donor-acceptor substrate ratio and improved LDFT titers to 1.1 g/L in 24 h (FIG. 6).

Lastly, balancing expression levels of the LDFT biosynthetic pathway genes (fkp, wbgL, and Hp3/4 ft) increased efficiency of LDFT production. Decreasing expression of fkp reduces excessive ATP and GTP consumption in GDP-fucose production, potentially relieving the metabolic burden of regenerating nucleotide cofactors (FIG. 7A). Decreasing expression of wbgL helps synchronize 2′-FL production with Hp3/4FT's slower turnover rate, streamlining 2′-FL towards LDFT production (FIG. 7A). Decreasing or delaying Hp3/4 ft expression causes build-up of 2′-FL, which is rapidly exported from the cell (FIG. 7B). It has been hypothesized that LacY is an importer for 2′-FL (Shin et al., 2020), but enhanced lacY expression was still insufficient for LDFT production from 2′-FL feeding (FIG. 10). Expression of additional heterologous importers may improve 2-FL transport. Fucosyllactose transporters have been identified in gut prebiotic Bifidobacterium species and are ideal candidates for screening in further studies to improve LDFT production (Sakanaka et al., 2019). Examples of such transporters include ABC transporters FL transporter-1 and FL transporter 2-from Bifidobacterium longum subsp. infantis ATCC 15697. FL transporter-1 is made up of the domains set forth in SEQ ID NOS:19-21 and transports 2′-fucosyllactose and 3-fucosyllactose, while FL transporter-2 is made up of the domains set forth in SEQ ID NOS:22-24 and transports 2′-FL, 3-FL, LDFT, and LNFP I.

Due to concerns about strain virulence for the production of bioactive compounds, the HMO production technologies can be translated to nonpathogenic generally-recognized-as-safe (GRAS) strains such as Bacillus subtilis, Corynebacterium glutamicum, and Saccharomyces cerevisiae (Becker et al., 2018; Kaspar et al., 2019; Lian et al., 2018). For example, lactose transporters can also be introduced into hosts such as C. glutamicum as described by Shen et al. (Microb Cell Fact (2019) 18:51). Expression of known FucU and LacZ homologes (e.g., B. subtilis homologs set forth in SEQ ID NO:25 and SEQ ID NO:26), can be reduced or eliminated as described above for E. coli. Alternatively, host cells such as S. cerevisiae which are not known to express FucU homologs would not require such modifications. Advancements in GRAS strains' synthetic biology toolbox such as genome editing, vector expression systems, and tuning of gene expression has improved their industrial application in producing nutraceuticals, food additives and biofuels. Some of these GRAS hosts also enable post-translational modification of enzymes and localization of proteins into organelles or on membranes. Development of GRAS HMO fucosylation systems would also forge production routes for other fucosylated compounds for pharmaceutical research.

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IV. Exemplary Embodiments

Exemplary embodiments provided in accordance with the presently disclosed subject matter include, but are not limited to, the claims and the following embodiments:

- 1. A recombinant cell for production of an oligosaccharide product, the recombinant cell comprising:
- a polynucleotide encoding a first glycosyltransferase polypeptide having a first substrate selectivity, and
- a polynucleotide encoding a second glycosyltransferase polypeptide having a second substrate selectivity.
- 2. The recombinant cell of embodiment 1, further comprising one or more polynucleotides selected from the group consisting of:
- a polynucleotide encoding a nucleotide sugar pyrophosphorylase polypeptide,
- a monosaccharide transporter polypeptide, and
- an oligosaccharide transporter polypeptide.
- 3. The recombinant cell of embodiment 1 or 2, for production of an oligosaccharide comprising two or more fucose moieties, comprising:
- a polynucleotide encoding a first fucosyltransferase polypeptide having a first substrate selectivity, and
- a polynucleotide encoding a second fucosyltransferase polypeptide having a second substrate selectivity;
- and optionally comprising one or more of:
- a polynucleotide encoding a nucleotide sugar pyrophosphorylase polypeptide,
- a polynucleotide encoding a lactose transporter polypeptide, and
- a polynucleotide encoding an L-fucose transporter polypeptide.
- 4. The recombinant cell of embodiment 2 or embodiment 3, wherein the nucleotide sugar pyrophosphorylase polypeptide is a bifunctional glycokinase and nucleotide sugar pyrophosphorylase polypeptide.
- 5. The recombinant cell of embodiment 3 or embodiment 4, wherein the first fucosyltransferase polypeptide is an α1-2-fucosyltransferase polypeptide.
- 6. The recombinant cell of embodiment 5, wherein the α1-2-fucosyltransferase polypeptide is an E. coli 0126 α1-2-fucosyltransferase WbgL polypeptide.
- 7. The recombinant cell of embodiment 5 or embodiment 6, wherein the α1-2-fucosyltransferase polypeptide is an E. coli O126 α1-2-fucosyltransferase (WbgL) polypeptide (GenBank: ABE98421.1), an H. mustelae 12198 α1-2-fucosyltransferase (Hm2FT) polypeptide (GenBank: CBG40460), an E. coli 0128:B12 α1-2-fucosyltransferase (WbsJ) polypeptide (GenBank: AA037698.1), an H. pylori UA1234 α1-2-fucosyltransferase (Hp2FTa) polypeptide (GenBank: AAD29863.1), or an H. pylori UA802 α1-2-fucosyltransferase (Hp2FTb) polypeptide (GenBank: AAC99764.1).
- 8. The recombinant cell of any one of embodiments 3-7, wherein the second fucosyltransferase polypeptide is an α1-3-fucosyltransferase polypeptide.
- 9. The recombinant cell of embodiment 8, wherein the α1-3-fucosyltransferase polypeptide is a truncated α1-3-fucosyltransferase polypeptide.
- 10. The recombinant cell of embodiment 8 or embodiment 9, wherein the α1-3-fucosyltransferase polypeptide is an H. pylori UA948 α1-3/4-fucosyltransferase (Hp3/4FT) polypeptide (GenBank: AAF35291.2), an H. pylori ATCC43504 α1-3-fucosyltransferase (Hp3FT) polypeptide (GenBank: AAB93985), an H. pylori J99 α1-3-fucosyltransferase (Hp3FT) polypeptide (GenBank: AAD06169.1, AAD06573.1), an H. pylori NCTC11637 α1-3-fucosyltransferase (Hp3FT) polypeptide (GenBank: AAB93985), a B. fragilis NCTC 9343 α1-3/4-fucosyltransferase polypeptide (GenBank: CAH09495.1), or an H. hepaticus ATCC 51449 Hh0072 polypeptide (GenBank: AAP76669.1).
- 11. The recombinant cell of any one of embodiments 2-10, wherein the nucleotide sugar pyrophosphorylase polypeptide is a B. fragilis bifunctional L-fucokinase/GDP-L-fucose pyrophosphorylase (Fkp) polypeptide.
- 12. The recombinant cell of any one of embodiments 3-11, which is transformed with a first expression vector comprising:
- the polynucleotide encoding the first fucosyltransferase polypeptide,
- the polynucleotide encoding the nucleotide sugar pyrophosphorylase polypeptide,
- the polynucleotide encoding the lactose transporter polypeptide, and
- the polynucleotide encoding the L-fucose transporter polypeptide.
- 13. The recombinant cell of any one of embodiments 3-12, wherein the polynucleotide encoding the first fucosyltransferase polypeptide and the polynucleotide encoding the nucleotide sugar pyrophosphorylase polypeptide are operably linked to a first inducible promoter.
- 14. The recombinant cell of embodiment 13, wherein the first inducible promoter is a P_LLacO1promoter.
- 15. The recombinant cell of any one of embodiments 3-14, wherein the polynucleotide encoding the second fucosyltransferase polypeptide is operably linked to a second inducible promoter.
- 16. The recombinant cell of embodiment 15, wherein the second inducible promoter is a P_LtetO1promoter.
- 17. The recombinant cell of any one of embodiments 3-16, wherein the L-fucose transporter polypeptide is an E. coli FucP polypeptide.
- 18. The recombinant cell of any one of embodiments 3-17, wherein the lactose transporter polypeptide is an E. coli LacY polypeptide.
- 19. The recombinant cell of any one of embodiments 3-18, wherein the polynucleotide encoding the lactose transporter polypeptide and the polynucleotide encoding the L-fucose transporter polypeptide are operably linked to a constitutive promoter.
- 20. The recombinant cell of any one of embodiments 1-19, which is modified to eliminate or reduce expression of an L-fucose mutarotase.
- 21. The recombinant cell of embodiment 20, wherein the L-fucose mutarotase is E. coli fucU.
- 22. The recombinant cell of any one of embodiments 1-21, which is modified to reduce or eliminate expression of a β-galactosidase.
- 23. The recombinant cell of embodiment 22, wherein the β-galactosidase is E. coli LacZ.
- 24. The recombinant cell of any one of embodiments 1-23, further comprising an polynucleotide encoding an additional transporter polypeptide.
- 25. The recombinant cell of embodiment 24, wherein the additional transporter polypeptide is a Bifidobacterium fucosyllactose transporter polypeptide.
- 26. The recombinant cell of any one of embodiments 1-25, which is an E. coli cell, a B. subtilis cell, a C. glutamicum cell, or an S. cerevisiae cell.
- 27. The recombinant cell of embodiment 26, which is an E. coli BW25113 Z1 cell or an E. coli MG1655 Z1 cell.
- 28. A method for producing an oligosaccharide product comprising two or more fucose moieties, the method comprising culturing a recombinant cell according to any one of embodiments 1-27 in a cell culture medium comprising L-fucose, an oligosaccharide acceptor, and a carbon source;
- wherein first glycosyltransferase is a first fucosyltransferase; the second glycosyltransferase is a second fucosyltransferase; and
- wherein the cell is cultured under conditions in which the first fucosyltransferase polypeptide, the second fucosyltransferase polypeptide, and the oligosaccharide acceptor is converted to the difucosylated oligosaccharide.
- 29. The method of embodiment 28, wherein the oligosaccharide transporter polypeptide is a lactose transporter polypeptide; the monosaccharide transporter polypeptide is an L-fucose transporter polypeptide; and the nucleotide sugar pyrophosphorylase polypeptide, the lactose transporter polypeptide, and the L-fucose transporter polypeptide are expressed under the culture conditions.
- 30. The method of embodiment 28 or embodiment 29, wherein the oligosaccharide acceptor is lactose and the oligosaccharide product is lactodifucotetraose (LDFT).
- 31. The method of any one of embodiments 28-29, wherein the carbon source comprises glucose, glycerol, or a combination thereof.
- 32. The method of any one of embodiments 28-31, wherein expression of the nucleotide sugar pyrophosphorylase polypeptide and the first fucosyltransferase polypeptide is induced at a level corresponding to 30-40% of maximum level (e.g., with isopropyl β-D-1-thiogalactopyranoside in an amount around 50 μM).
- 33. The method of embodiment 32, wherein expression of the second fucosyltransferase polypeptide is induced at a maximum level (e.g., with anhydrotetracycline at around 100 ng/mL).

Although the foregoing has been described in some detail by way of illustration and example for purposes of clarity and understanding, one of skill in the art will appreciate that certain changes and modifications can be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference.

V. Informal Sequence Listing

SEQ ID NO: 1. E. coli 0126 α1-2-fucosyltransferase (WbgL) (GenBank: ABE98421.1)

1
MSIIRLQGGL GNQLFQFSFG YALSKINGTP LYFDISHYAE NDDHGGYRLN NLQIPEEYLQ

61
YYTPKINNIY KLLVRGSRLY PDIFLFLGFC NEFHAYGYDF EYIAQKWKSK KYIGYWQSEH

121
FFHKHILDLK EFFIPKNVSE QANLLAAKIL ESQSSLSIHI RRGDYIKNKT ATLTHGVCSL

181
EYYKKALNKI RDLAMIRDVF IFSDDIFWCK ENIETLLSKK YNIYYSEDLS QEEDLWLMSL

241
ANHHIIANSS FSWWGAYLGS SASQIVIYPT PWYDITPKNT YIPIVNHWIN VDKHSSC

SEQ ID NO: 2. H. pylori UA948 α1-3/4-fucosyltransferase (Hp3/4FT) (GenBank:

AAF35291.2),

1
MFQPLLDAFI DSTHLDETTH KPPLNVALAN WWPLKNSEKK GERDFILHFI LKQRYKIILH

61
SNPNEPSDLV FGNPLEQARK ILSYQNTKRV FYTGENEVPN FNLEDYAIGF DELDENDRYL

121
RMPLYYAYLH YKAMLVNDTT SPYKLKALYT LKKPSHKFKE NHPNLCALIH NESDPWKRGE

181
ASFVASNPNA PIRNAFYDAL NAIEPVASGG SVKNTLGYKV KNKNEFLSQY KENLCFENSQ

241
GYGYVTEKIL DAYFSHTIPI YWGSPSVAKD FNPKSFVNVH DENNEDEAID YIRYLHAHQN

301
AYLDMLYENP LNTIDGKAGF YQDLSFEKIL DFFKNILEND TIYHCNDAHY SALHRDLNEP

361
LVSVDDLRRD HDDLRVNYDD LRVNYDDLRV NYDDLRVNYD DLRVNYDDLR RDHDDLRRDH

421
ERLLSKATPL LELSQNTSFK IYRKAYQKSL PLLRAIRRWV RK

SEQ ID NO: 3. Bacteroides fragilis bifunctional L fucokinase/GDP-L-fucose

pyrophosphorylase (BfFKP) (GenBank: CAH08307.1)

1
MQKLLSLPSN LVQSFHELER VNRTDWFCTS DPVGKKLGSG GGTSWLLEEC YNEYSDGATE

61
GEWLEKEKRI LLHAGGQSRR LPGYAPSGKI LTPVPVERWE RGQHLGQNLL SLQLPLYEKI

121
MSLAPDKLHT LIASGDVYIR SEKPLQSIPE ADVVCYGLWV DPSLATHHGV FASDRKHPEQ

181
LDEMLQKPSL AELESLSKTH LFLMDIGIWL LSDRAVEILM KRSHKESSEE LKYYDLYSDF

241
GLALGTHPRI EDEEVNTLSV AILPLPGGEF YHYGTSKELI SSTLSVQNKV YDQRRIMHRK

301
VKPNPAMFVQ NAVVRIPLCA ENADLWIENS HIGPKWKIAS RHIITGVPEN DWSLAVPAGV

361
CVDVVPMGDK GFVARPYGLD DVFKGDLRDS KTTLTGIPFG EWMSKRGLSY TDLKGRTDDL

421
QAVSVFPMVN SVEELGLVLR WMLSEPELEE GKNIWLRSEH FSADEISAGA NLKRLYAQRE

481
EFRKGNWKAL AVNHEKSVFY QLDLADAAED FVRLGLDMPE LLPEDALQMS RIHNRMLRAR

541
ILKLDGKDYR PEEQAAFDLL RDGLLDGISN RKSTPKLDVY SDQIVWGRSP VRIDMAGGWT

601
DTPPYSLYSG GNVVNLAIEL NGQPPLQVYV KPCKDFHIVL RSIDMGAMEI VSTFDELQDY

661
KKIGSPFSIP KAALSLAGFA PAFSAVSYAS LEEQLKDEGA GIEVTLLAAI PAGSGLGTSS

721
ILASTVLGAI NDFCGLAWDK NEICQRTLVL EQLLTTGGGW QDQYGGVLQG VKLLQTEAGF

781
AQSPLVRWLP DHLFTHPEYK DCHLLYYTGI TRTAKGILAE IVSSMELNSS LHLNLLSEMK

841
AHALDMNEAI QRGSFVEFGR LVGKTWEQNK ALDSGTNPPA VEAIIDLIKD YTLGYKLPGA

901
GGGGYLYMVA KDPQAAVRIR KILTENAPNP RARFVEMTLS DKGFQVSRS

SEQ ID NO: 4. E. coli str. K-12 substr. MG1655 LacY (GenBank: AAC73446.1)

1
MYYLKNTNFW MFGLFFFFYF FIMGAYFPFF PIWLHDINHI SKSDTGIIFA AISLESLLFQ

61
PLFGLLSDKL GLRKYLLWII TGMLVMFAPF FIFIFGPLLQ YNILVGSIVG GIYLGFCENA

121
GAPAVEAFIE KVSRRSNFEF GRARMFGCVG WALCASIVGI MFTINNQFVF WLGSGCALIL

181
AVLLFFAKTD APSSATVANA VGANHSAFSL KLALELFRQP KLWFLSLYVI GVSCTYDVED

241
QQFANFFTSF FATGEQGTRV FGYVTTMGEL LNASIMFFAP LIINRIGGKN ALLLAGTIMS

301
VRIIGSSFAT SALEVVILKT LHMFEVPFLL VGCFKYITSQ FEVRESATIY LVCFCFFKQL

361
AMIFMSVLAG NMYESIGFQG AYLVLGLVAL GFTLISVFTL SGPGPLSLLR RQVNEVA

SEQ ID NO: 5. E. coli K-12 substr. MG1655 FucP (AAC75843.1)

1
MGNTSIQTQS YRAVDKDAGQ SRSYIIPFAL LCSLFFLWAV ANNLNDILLP QFQQAFTLTN

61
FQAGLIQSAF YFGYFIIPIP AGILMKKLSY KAGIITGLFL YALGAALFWP AAEIMNYTLE

121
LVGLFIIAAG LGCLETAANP FVTVLGPESS GHERLNLAQT FNSFGAIIAV VFGQSLILSN

181
VPHQSQDVLD KMSPEQLSAY KHSLVLSVQT PYMIIVAIVL LVALLIMLTK FPALQSDNHS

241
DAKQGSFSAS LSRLARIRHW RWAVLAQFCY VGAQTACWSY LIRYAVEEIP GMTAGFAANY

301
LTGTMVCFFI GRFTGTWLIS RFAPHKVLAA YALIAMALCL ISAFAGGHVG LIALTLCSAF

361
MSIQYPTIFS LGIKNLGQDT KYGSSFIVMT IIGGGIVTPV MGFVSDAAGN IPTAELIPAL

421
CFAVIFIFAR FRSQTATN

SEQ ID NO: 6. E. coli K-12 substr. MG1655 FucU (AAC75846.1)

1
MLKTISPLIS PELLKVLAEM GHGDEIIFSD AHFPAHSMGP QVIRADGLLV SDLLQAIIPL

61
FELDSYAPPL VMMAAVEGDT LDPEVERRYR NALSLQAPCP DIIRINRFAF YERAQKAFAI

121
VITGERAKYG NILLKKGVTP

SEQ ID NO: 7. E. coli LacZ K-12 substr. MG1655 (AAC73447.1)

1
MTMITDSLAV VLQRRDWENP GVTQLNRLAA HPPFASWRNS EEARTDRPSQ QLRSLNGEWR

61
FAWFPAPEAV PESWLECDLP EADTVVVPSN WQMHGYDAPI YTNVTYPITV NPPFVPTENP

121
TGCYSLTFNV DESWLQEGQT RIIFDGVNSA FHLWCNGRWV GYGQDSRLPS EFDLSAFLRA

181
GENRLAVMVL RWSDGSYLED QDMWRMSGIF RDVSLLHKPT TQISDEHVAT RENDDESRAV

241
LEAEVQMCGE LRDYLRVTVS LWQGETQVAS GTAPFGGEII DERGGYADRV TLRLNVENPK

301
LWSAEIPNLY RAVVELHTAD GTLIEAEACD VGFREVRIEN GLLLLNGKPL LIRGVNRHEH

361
HPLHGQVMDE QTMVQDILLM KQNNENAVRC SHYPNHPLWY TLCDRYGLYV VDEANIETHG

421
MVPMNRLTDD PRWLPAMSER VTRMVQRDRN HPSVIIWSLG NESGHGANHD ALYRWIKSVD

481
PSRPVQYEGG GADTTATDII CPMYARVDED QPFPAVPKWS IKKWLSLPGE TRPLILCEYA

541
HAMGNSLGGF AKYWQAFRQY PRLQGGFVWD WVDQSLIKYD ENGNPWSAYG GDFGDTPNDR

601
QFCMNGLVFA DRTPHPALTE AKHQQQFFQF RLSGQTIEVT SEYLFRHSDN ELLHWMVALD

661
GKPLASGEVP LDVAPQGKQL IELPELPQPE SAGQLWLTVR VVQPNATAWS EAGHISAWQQ

721
WRLAENLSVT LPAASHAIPH LTTSEMDFCI ELGNKRWQFN RQSGFLSQMW IGDKKQLLTP

781
LRDQFTRAPL DNDIGVSEAT RIDPNAWVER WKAAGHYQAE AALLQCTADT LADAVLITTA

841
HAWQHQGKTL FISRKTYRID GSGQMAITVD VEVASDTPHP ARIGLNCQLA QVAERVNWLG

901
LGPQENYPDR LTAACFDRWD LPLSDMYTPY VFPSENGLRC GTRELNYGPH QWRGDFQFNI

961
SRYSQQQLME TSHRHLLHAE EGTWLNIDGF HMGIGGDDSW SPSVSAEFQL SAGRYHYQLV

1021
WCQK

SEQ ID NO: 8. H. mustelae 12198 α1-2-fucosyltransferase (Hm2FT) (GenBank:

CBG40460),

1
MDFKIVQVHG GLGNQMFQYA FAKSLQTHLN IPVLLDTTWF DYGNRELGLH LEPIDLQCAS

61
AQQIAAAHMQ NLPRLVRGAL RRMGLGRVSK EIVFEYMPEL FEPSRIAYFH GYFQDPRYFE

121
DISPLIKQTF TLPHPTEHAE QYSRKLSQIL AAKNSVFVHI RRGDYMRLGW QLDISYQLRA

181
IAYMAKRVQN LELFLFCEDL EFVQNLDLGY PFVDMTTRDG AAHWDMMLMQ SCKHGIITNS

241
TYSWWAAYLI KNPEKIIIGP SHWIYGNENI LCKDWVKIES QFETKS

SEQ ID NO: 9. E. coli 0128:B12 α1-2-fucosyltransferase (WbsJ) (GenBank: AAO37698.1),

1
MEVKIIGGLG NQMFQYATAF AIAKRTHQNL TVDISDAVKY KTHPLRLVEL SCSSEFVKKA

61
WPFEKYLESE KIPHEMKKGM FRKHYVEKSL EYDPDIDTKS INKKIVGYFQ TEKYFKEFRH

121
ELIKEFQPKT KENSYQNELL NLIKENDTCS LHIRRGDYVS SKIANETHGT CSEKYFERAI

181
DYLMNKGVIN KKTLLFIFSD DIKWCRENIF FNNQICFVQG DAYHVELDML LMSKCKNNII

241
SNSSFSWWAA WLNENKNKTV IAPSKWFKKD IKHDIIPESW VKL

SEQ ID NO: 10. H. pylori UA1234 α1-2-fucosyltransferase (Hp2FTa) (GenBank:

AAD29863.1),

1
MAFKVVQICG GLGNQMFQYA FAKSLQKHSN TPVLLDITSF DWSNRKMQLE LFPIDLPYAS

61
EKEIAIAKMQ HLPKLVRNVL KCMGEDRVSQ EIVFEYEPKL LKTSRLTYFY GYFQDPRYED

121
AISPLIKQTF TLPPPPENGN NKKKEEEYHR KLALILAAKN SVEVHIRRGD YVGIGCQLGI

181
DYQKKALEYM AKRVPNMELF VFCEDLEFTQ NLDLGYPEMD MTTRDKEEEA YWDMLLMQSC

241
KHGIIANSTY SWWAAYLINN PEKIIIGPKH WLFGHENILC KEWVKIESHF EVKSQKYNA

SEQ ID NO: 11. H. pylori UA802 α1-2-fucosyltransferase (Hp2FTb) (GenBank:

AAC99764.1).

1
MAFKVVQICG GLGNQMFQYA FAKSLQKHLN TPVLLDTTSF DWSNRKMQLE LFPIDLPYAN

61
AKEIAIAKMQ HLPKLVRDAL KYIGFDRVSQ EIVFEYEPKL LKPSRLTYFF GYFQDPRYED

121
AISSLIKQTF TLPPPPENNK NNNKKEEEYQ RKLSLILAAK NSVFVHIRRG DYVGIGCQLG

181
IDYQKKALEY MAKRVPNMEL FVFCEDLKFT QNLDLGYPFT DMTTRDKEEE AYWDMLLMQS

241
CKHGIIANST YSWWAAYLME NPEKIIIGPK HWLFGHENIL CKEWVKIESH FEVKSQKYNA

SEQ ID NO: 12. H. pylori ATCC43504 α1-3-fucosyltransferase (Hp43504 3FT) (GenBank:

AAB93985),

1
MPLYYDRLHH KAESVNDTTA PYKIKGNSLY TLKKPSHCFK ENHPNLCALI NNESDPLKRG

61
FASEVASNAN APMRNAFYDA LNSIEPVTGG GAVKNTLGYK VGNKSEFLSQ YKENLCFENS

121
QGYGYVTEKI IDAYFSHTIP IYWGSPSVAK DENPKSFVNV HDENNEDEAI DYVRYLHTHP

181
NAYLDMLYEN PLNTLDGKAY FYQNLSFKKI LDFFKTILEN DTIYHNNPFI FYRDLNEPLV

241
SIDNLRINYD NLRVNYDDLR VNYDDLRVNY DDLRINYDDL RINYDDLRIN YERLLQNASP

301
LLELSQNTSF KIYRKIYQKS LPLLRVIRRW VKK

SEQ ID NO: 13. H. pylori J99 α1-3-fucosyltransferase (HpJ99 3FT) (GenBank:

AAD06169.1),

1
MFQPLLDAYT DSTRLDETDY KPPLNIALAN WWPLDKRESK GFRRFILYFI LSQRYTITLH

61
QNPNEPSDLV FGSPIGSARK ILSYQNTKRV FYTGENEVPN FNLFDYAIGF DELDERDRYL

121
RMPLYYASLH YKAESVNDTT APYKLKDNSL YALKKPSHHF KENHPNLCAV VNDESDPLKR

181
GFASFVASNP NAPIRNAFYD ALNSIEPVTG GGSVKNTLGY NVKNKSEFLS QYKENLCFEN

241
TQGYGYVTEK IIDAYFSHTI PIYWGSPSVA KDENPKSFVN VCDFKNFDEA IDYVRYLHTH

301
PNAYLDMLYE NPLNTLDGKA YFYQNLSFKK ILDFFKTILE NDTIYHDNPF IFYRDLNEPL

361
VAIDDLRVNY DDLRVNYDDL RVNYDDLRVN YDDLRVNYDD LRVNYDDLRV NYDRLLQNAS

420
PLLELSQNTT FKIYRKAYQK SLPLLRTIRR WVKK

SEQ ID NO: 14. H. pylori J99 α1-3-fucosyltransferase (HpJ99 3FT) (GenBank:

AAD06573.1),

1
MFQPLLDAFI ESTPIKKKIT FKSPPPPLKI AVANWWGGAE EFKKSTLYFI LSQRYTITLH

61
QNPNEPSDLV LGSPIGSARK ILSYQNTKRV FYTGENEVPN FNLFDYAIGF DELDERDRYL

121
RMPLYYASLH YKAESVNDTT APYKLKDNSL YALKKPSHHF KENHPNLCAV VNDESDPLKR

181
GFASEVASNP NAPIRNAFYD ALNSIEPVTG GGSVKNTLGY NVKNKSEFLS QYKENLCFEN

241
TQGYGYVTEK IIDAYFSHTI PIYWGSPSVA KDENPKSFVN VCDFKNFDEA IDYVRYLHTH

301
PNAYLDMLYE NPLNTLDGKA YFYQNLSFKK ILDFFKTILE NDTIYHDNPF IFYRDLNEPL

361
VAIDDLRVNY DDLRVNYDDL RVNYDDLRVN YDRLLQNASP LLELSQNTTF KIYRKAYQKS

421
LPLLRAIRRW VKKLGL

SEQ ID NO: 15. H. pylori NCTC11637 α1-3-fucosyltransferase (Hp11637 3FT) (GenBank:

AAB93985).

1
MPLYYDRLHH KAESVNDTTA PYKIKGNSLY TLKKPSHCFK ENHPNLCALI NNESDPLKRG

61
FASFVASNAN APMRNAFYDA LNSIEPVTGG GAVKNTLGYK VGNKSEFLSQ YKENLCFENS

121
QGYGYVTEKI IDAYFSHTIP IYWGSPSVAK DENPKSFVNV HDENNEDEAI DYVRYLHTHP

181
NAYLDMLYEN PLNTLDGKAY FYQNLSFKKI LDFFKTILEN DTIYHNNPFI FYRDLNEPLV

241
SIDNLRINYD NLRVNYDDLR VNYDDLRVNY DDLRINYDDL RINYDDLRIN YERLLQNASP

301
LLELSQNTSF KIYRKIYQKS LPLLRVIRRW VKK

SEQ ID NO: 16. B. fragilis NCTC 9343 α1-3/α1-4-fucosyltransferase (Bf3/4ft) (GenBank:

CAH09495.1)

1
MDILILFYNT MWGFPLEFRK EDLPGGCVIT TDRNLIAKAD AVVFHLPDLP SVMEDEIDKR

61
EGQLWVGWSL ECEENYSWTK DPEFRESEDL WMGYHQEDDI VYPYYGPDYG KMLVTARREK

121
PYKKKACMFI SSDMNRSHRQ EYLKELMQYT DIDSYGKLYR NCELPVEDRG RDTLLSVIGD

181
YQFVISFENA IGKDYVTEKF FNPLLAGTVP VYLGAPNIRE FAPGENCFLD ICTFDSPEGV

241
AAFMNQCYDD EALYERFYAW RKRPLLLSFT NKLEQVRSNP LIRLCQKIHE LKLGGI

SEQ ID NO: 17. H. hepaticus ATCC 51449 Hh0072 (GenBank: AAP76669.1)

1
MKDDLVILHP DGGIASQIAF VALGLAFEQK GAKVKYDLSW FAEGAKGEWN PSNGYDKVYD

61
ITWDISKAFP ALHIEIANEE EIERYKSKYL IDNDRVIDYA PPLYCYGYKG RIFHYLYAPF

121
FAQSFAPKEA QDSHTPFAAL LQEIESSPSP CGVHIRRGDL SQPHIVYGNP TSNEYFAKSI

181
ELMCLLHPQS SFYLESDDLA FVKEQIVPLL KGKTYRICDV NNPSQGYLDL YLLSRCRNII

241
GSQGSMGEFA KVLSPHNPLL ITPRYRNIFK EVENVMCVNW GESVQHPPLV CSAPPPLVSQ

301
LKRNAPLNSR LYKEKDNASA

SEQ ID NO: 18. A. thaliana FKGP (UniProt: Q9LNJ9)

1
MSKQRKKADL ATVLRKSWYH LRLSVRHPTR VPTWDAIVLT AASPEQAELY DWQLRRAKRM

61
GRIASSTVTL AVPDPDGKRI GSGAATLNAI YALARHYEKL GFDLGPEMEV ANGACKWVRF

121
ISAKHVLMLH AGGDSKRVPW ANPMGKVFLP LPYLAADDPD GPVPLLEDHI LAIASCARQA

181
FQDQGGLFIM TGDVLPCFDA FKMTLPEDAA SIVTVPITLD IASNHGVIVT SKSESLAESY

241
TVSLVNDLLQ KPTVEDLVKK DAILHDGRTL LDTGIISARG RAWSDLVALG CSCQPMILEL

301
IGSKKEMSLY EDLVAAWVPS RHDWLRTRPL GELLVNSLGR QKMYSYCTYD LQFLHFGTSS

361
EVLDHLSGDA SGIVGRRHLC SIPATTVSDI AASSVILSSE IAPGVSIGED SLIYDSTVSG

421
AVQIGSQSIV VGIHIPSEDL GTPESFREML PDRHCLWEVP LVGHKGRVIV YCGLHDNPKN

481
SIHKDGTFCG KPLEKVLFDL GIEESDLWSS YVAQDRCLWN AKLFPILTYS EMLKLASWLM

541
GLDDSRNKEK IKLWRSSQRV SLEELHGSIN FPEMCNGSSN HQADLAGGIA KACMNYGMLG

601
RNLSQLCHEI LQKESLGLEI CKNFLDQCPK FQEQNSKILP KSRAYQVEVD LLRACGDEAK

661
AIELEHKVWG AVAEETASAV RYGFREHLLE SSGKSHSENH ISHPDRVFQP RRTKVELPVR

721
VDFVGGWSDT PPWSLERAGY VLNMAITLEG SLPIGTIIET TNQMGISIQD DAGNELHIED

781
PISIKTPFEV NDPFRLVKSA LLVTGIVQEN FVDSTGLAIK TWANVPRGSG LGTSSILAAA

841
VVKGLLQISN GDESNENIAR LVLVLEQLMG TGGGWQDQIG GLYPGIKFTS SFPGIPMRLQ

901
VVPLLASPQL ISELEQRLLV VFTGQVRLAH QVLHKVVTRY LQRDNLLISS IKRLTELAKS

SEQ ID NO: 19. B. longum subsp. infantis ATCC 15697 FL transporter-1 domain Blon-0341

(ACJ51465.1)

1
MTNATAQPDT SVMRKPKRQY IGILYCLPYV VVFLFGMIVP MFYALYLSFF KQSLLGGTTE

61
AGFDNFIRAF KDEALWGGFR NVLIYAAIQI PMNLILSLVA ALVLDSQRIR HIAVPRILLF

121
LPYAVPGVIA ALMWGYIYGD KYGLFGQIAG MFGVAAPNML SKQLMLFAIA NICTWCFLGY

181
NMLIYYSALI GIPNDLYESA RIDGASELRI AWSVKIPQIK STIVMTVLES VIGTLQLENE

241
PNILRTSAPD VINSSYTPNI YTYNLAFNGQ NVNYAAAVSL VIGIIVMALV AVVKIIGNKW

301
ENK

SEQ ID NO: 20. B. longum subsp. infantis ATCC 15697 FL transporter-1 domain Blon-0342

(ACJ51466.1)

1
MSEAIARPRS KSLQRRDAKL ALKASKHYKR MQQREPAPKL TGKQRVLNWL LHIIMAVMVI

61
YCLVPLLWVV FSSTKTSEGI FSSFGLWEDD KNVEWQNVQD TFAYQHGVYT RWLENTIMYA

121
VVAGVGATII ATFAGYAIAT MRFPGRNALL AVTLAFMSIP STVITVPLFL MYSKIGLVGT

181
PWAVIIPQLA TPFGLYLMII YAQTSIPVSL IEAAKLDGAN TWTIFWKVGF PLLSPGFVTV

241
LLFTLVGVWN NYFLPLIMLT NTNDYPLTVG LNMWLKMGAQ GTSDGQVPNN LIITGSLIAV

301
VPLIIAFMFL QKYWQSGLAA GSVKQ

SEQ ID NO: 21. B. longum subsp. infantis ATCC 15697 FL transporter-1 domain Blon-0343

(ACJ51467.1)

1
MTHKGVIMKK SIRLIAAVAA LAMTAGAAAC GSGTSQKNNK ADVSLNDINS ALTDTSKTTD

61
LTVWAYSAKQ IEGPVKAFQE RYPHIKINFV NTGAASDHFT KFQNVVSANK GVPDVVQMSI

121
SEYEQYAVSG ALLNFESDEI EKAWGTQYAQ AAWKNVHFGG GLYGTPQDAA PLALYVRKDI

181
LDEHGLKVPT TWQEFYDEGV KLHKQDPSKY MGFISSSDTS LFGVLRTVGA KPWTVKDTTN

241
IDFSLTTGRV AEFIKFIQKC LDDGVLRAAA TGTDEFNREV NDGVYATRLE GCWQGNIYKD

301
QNPSLKGKMV VAHPLAWGND GESYQSESTG SMFSVSSATP KDKQAAALAF IQWVNGSKDG

361
VSEFLTANKG NYFMASNYYQ KDKSKRDQQE TDGYFANTNV NEIYFESMDK VNMDWDYIPF

421
PAQLTVAFGD TVAPALTGKG DLLTAFTKLQ DNLKSYAEDN GFKVTTDAD

SEQ ID NO: 22. B. longum subsp. infantis ATCC 15697 FL transporter-2 domain Blon-2202

(ACJ51465.1)

1
MKKSIRLVAA IAALAMTAGI SACGSSTNGN QAKSDVTAQD VENALTDTSK NVELTVWAYS

61
AKQMEPTVKA FEKKYPHIKI NFVNTGAAED HFTKFQNVVQ AQKDIPDVVQ MSANKFQQFA

121
VSGALLNFAN DSIEKAWSKL YTKTAWAQVH YAGGLYGAPQ DATPLANYVR KDILDEHNLQ

181
VPESWEDIYN EGIKLHKEDS NKYMGILGSD ISFFTNLYRS VGARLWKVNS VDDVELTMNS

241
GKAKEFTEFL QKCLKDGVLE GGTVFTDEFN RSINDGRYAT FINENWMGNT YKEQNPSLKG

301
KMVVAAPPSW KGQPYQSSSV GSMMSVSAAC PKEKQAAALA FINWLDSDKD AIQSWQDTNN

361
GNFFMAASVY QDDENQRNKK ETDGYYANDD VNAVYFDSMD KVNTDWEYLP FMSQVEVVEN

421
DVIVPEMNEN GDLVGAMAKA QQKLKAYAED NGFKVTTDAD

SEQ ID NO: 23. B. longum subsp. infantis ATCC 15697 FL transporter-2 domain Blon-2203

(ACJ53263.1)

1
MSEAIARPRS KSLQRRDAKL ALKASKHYKR MQQREPAPKL TGKQRVLNWL LHIIMAVMVI

61
YCLVPLLWVV FSSTKTSEGI FSSFGLWEDD KNVFWQNVQD TFAYQHGVYT RWLENTIMYA

121
VVAGVGATII ATFAGYAIAT MRFPGRNALL AVTLAFMSIP STVITVPLEL MYSKIGLVGT

181
PWAVIIPQLA TPFGLYLMII YAQTSIPVSL IEAAKLDGAN TWTIFWKVGF PLLSPGFVTV

241
LLFTLVGVWN NYFLPLIMLT NTNDYPLTVG LNMWLKMGAQ GTSDGQVPNN LIITGSLIAV

301
VPLIIAFMFL QKYWQSGLAA GSVKQ

SEQ ID NO: 24. B. longum subsp. infantis ATCC 15697 FL transporter-2 domain Blon-2204

(ACJ53264.1)

1
MTNATAQPDT SVMRKPKRQY IGILYCLPYV VVFLFGMIVP MFYALYLSFF KQSLLGGTTF

61
AGFDNFIRAF KDEALWGGFR NVLIYAAIQI PMNLILSLVA ALVLDSQRIR HIAVPRILLE

121
LPYAVPGVIA ALMWGYIYGD KYGLFGQIAG MFGVAAPNML SKQLMLFAIA NICTWCFLGY

181
NMLIYYSALI GIPNDLYESA RIDGASELRI AWSVKIPQIK STIVMTVLES VIGTLQLENE

241
PNILRTSAPD VINSSYTPNI YTYNLAFNGQ NVNYAAAVSL VIGIIVMALV AVVKIIGNKW

301
ENK

SEQ ID NO: 25. B. subtilus FucU homolog (WP_158321581.1)

1
MLKGIPAILS PDLMKVLMEM GHGDEIVLAD GNFPSASHAQ NLLRCDGHGI PALLEAILKE

61
FPLDTYVEHP VTLMDVVEGE QFQPTIWQDF EKVIQKEHGP ALQMEYLDRF TFYERAKKAY

121
AIVATGEAAQ YANIILKKGV VK

SEQ ID NO: 26. B. subtilus LacZ homolog (MBA5241670.1)

1
MEVTDVRLRV DRENPGVTQL NRLAAHPPFA SWRNSEEART DRPSQQLRSL NGEWRFAWFP

61
APEAVPESWL ECDLPEADTV VVPSNWQMHG YDAPIYTNVT YPITVNPPFV PTENPTGCYS

121
LTFNVDESWL QEGQTRIIFD GVNSAFHLWC NGRWVGYGQD SRLPSEFDLS AFLRAGENRL

181
AVMVLRWSDG SYLEDQDMWR MSGIFRDVSL LHKPTTQISD FHVATRENDD FSRAVLEAEV

241
QMCGELRDYL RVTVSLWQGE TQVASGTAPF GGEIIDERGG YADRVTLRLN VENPKLWSAE

301
IPNLYRAVVE LHTADGTLIE AEACDVGFRE VRIENGLLLL NGKPLLIRGV NRHEHHPLHG

361
QVMDEQTMVQ DILLMKQNNF NAVRCSHYPN HPLWYTLCDR YGLYVVDEAN IETHGMVPMN

421
RLTDDPRWLP AMSERVTRMV QRDRNHPSVI IWSLGNESGH GANHDALYRW IKSVDPSRPV

481
QYEGGGADTT ATDIICPMYA RVDEDQPFPA VPKWSIKKWL SLPGETRPLI LCEYAHAMGN

541
SLGGFAKYWQ AFRQYPRLQG GFVWDWVDQS LIKYDENGNP WSAYGGDFGD TPNDRQFCMN

601
GLVFADRTPH PALTEAKHQQ QFFQFRLSGQ TIEVTSEYLF RHSDNELLHW MVALDGKPLA

661
SGEVPLDVAP QGKQLIELPE LPQPESAGQL WLTVRVVQPN ATAWSEAGHI SAWQQWRLAE

721
NLSVTLPAAS HAIPHLTTSE MDFCIELGNK RWQFNRQSGF LSQMWIGDKK QLLTPLRDQF

781
TRAPLDNDIG VSEATRIDPN AWVERWKAAG HYQAEAALLQ CTADTLADAV LITTAHAWQH

841
QGKTLFISRK TYRIDGSGQM AITVDVEVAS DTPHPARIGL NCQLAQVAER VNWLGLGPQE

901
NYPDRLTAAC FDRWDLPLSD MYTPYVEPSE NGLRCGTREL NYGPHQWRGD FQFNISRYSQ

961
QQLMETSHRH LLHAEEGTWL NIDGFHMGIG GDDSWSPSVS AELQLSAGRY HYQLVWCQK

	Number	Date	Country
Parent	PCT/US2022/017074	Feb 2022	US
Child	18450181		US

METHODS FOR PRODUCTION OF FUCOSYLATED OLIGOSACCHARIDES IN RECOMBINANT CELL CULTURE

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Original Assignees

CPC

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Abstract

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CROSS-REFERENCES TO RELATED APPLICATIONS

Provisional Applications (1)

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